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Human kynurenine aminotransferase I/glutaminetransaminase K (hKAT-I) is an important multifunctional enzyme. This study systematically studies the substrates of hKAT-I and reassesses the effects of pH, Tris, amino acids andα-keto acids on the activity of the enzyme. The experiments were comprised of functional expression of the hKAT-I in an insect cell/baculovirus expression system, purification of its recombinant protein, and functional characterization of the purified enzyme. This study demonstrates that hKAT-I can catalyze kynurenine to kynurenic acid under physiological pH conditions, indicates indo-3-pyruvate and cysteine as efficient inhibitors for hKAT-I, and also provides biochemical information about the substrate specificity and cosubstrate inhibition of the enzyme. hKAT-I is inhibited by Tris under physiological pH conditions, which explains why it has been concluded that the enzyme could not efficiently catalyze kynurenine transamination. Our findings provide a biochemical basis towards understanding the overall physiological role of hKAT-Iin vivoand insight into controlling the levels of endogenous kynurenic acid through modulation of the enzyme in the human brain. [ABSTRACT FROM AUTHOR]

Bacterial RNA polymerase (RNAP) is the central enzyme of gene expression that is responsible for the synthesis of all types of cellular RNAs. The process of transcription is accompanied by complex structural rearrangements of RNAP. Despite the recent progress in structural studies of RNAP, detailed mechanisms of conformational changes of RNAP that occur at different stages of transcription remain unknown. The goal of this work was to obtain novel ligands to RNAP which would target different epitopes of the enzyme and serve as specific probes to study the mechanism of transcription and conformational flexibility of RNAP. Usingin vitroselection methods, we obtained 13 classes of ssDNA aptamers againstEscherichia colicore RNAP. The minimal nucleic acid scaffold (an oligonucleotide construct imitating DNA and RNA in elongation complex), rifampicin and theσ70-subunit inhibited binding of the aptamers to RNAP core but did not affect the dissociation rate of preformed RNAP–aptamer complexes. We argue that these ligands sterically block access of the aptamers to their binding sites within the main RNAP channel. In contrast, transcript cleavage factor GreB increased the rate of dissociation of preformed RNAP–aptamer complexes. This suggested that GreB that binds RNAP outside the main channel actively disrupts RNAP–aptamer complexes by inducing conformational changes in the channel. We propose that the aptamers obtained in this work will be useful for studying the interactions of RNAP with various ligands and regulatory factors and for investigating the conformational flexibility of the enzyme. [ABSTRACT FROM AUTHOR]

The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungusTalaromyces emersonii, PDB 1Q9H, was solved to 2.4 Å by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of≈ 60 Å × 40 Å × 50 Å and an overallβ-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A fromTrichoderma reeseiis conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties ofT. emersonii1Q9H are different. The gene (cel7) encoding CBH IB was isolated fromT. emersoniiand expressed heterologously with an N-terminal polyHis-tag, inEscherichia coli. The deduced amino acid sequence ofcel7is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles ofcel7expression inT. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of thecel7genomic sequence. [ABSTRACT FROM AUTHOR]

The classical metabolic control theory[Kacser, H.&Burns, J.A. (1973)Symp. Soc. Exp. Biol.27, 65–104; Heinrich, R.&Rapoport, T. (1974)Eur. J. Biochem.42, 89–95.] does not take into account experimental evidence for correlations between enzyme concentrations in the cell. We investigated the implications of two causes of linear correlations: competition between enzymes, which is a mere physical adaptation of the cell to the limitation of resources and space, and regulatory correlations, which result from the existence of regulatory networks. These correlations generate redistribution of enzyme concentrations when the concentration of an enzyme varies; this may dramatically alter the flux and metabolite concentration curves. In particular, negative correlations cause the flux to have a maximum value for a defined distribution of enzyme concentrations. Redistribution coefficients of enzyme concentrations allowed us to calculate the‘combined response coefficient’ that quantifies the response of flux or metabolite concentration to a perturbation of enzyme concentration. [ABSTRACT FROM AUTHOR]

Aspergillus nigerisopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure ofPenicillium minioluteumdextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and theKm value was sixfold higher and thek0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite−1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal andβ-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzymes promote a number of oxidative biotransformations including the hydroxylation of unactivated hydrocarbons. Whereas the long-standing consensus view of the P450 mechanism implicates a high-valent iron-oxene species as the predominant oxidant in the radicalar hydrogen abstraction/oxygen rebound pathway, more recent studies on isotope partitioning, product rearrangements with‘radical clocks’, and the impact of threonine mutagenesis in P450s on hydroxylation rates support the notion of the nucleophilic and/or electrophilic (hydro)peroxo-iron intermediate(s) to be operative in P450 catalysis in addition to the electrophilic oxenoid-iron entity; this may contribute to the remarkable versatility of P450s in substrate modification. Precedent to this mechanistic concept is given by studies with natural and synthetic P450 biomimics. While the concept of an alternative electrophilic oxidant necessitates C-H hydroxylation to be brought about by a cationic insertion process, recent calculations employing density functional theory favour a‘two-state reactivity’ scenario, implicating the usual ferryl-dependent oxygen rebound pathway to proceed via two spin states (doublet and quartet); state crossing is thought to be associated with either an insertion or a radicalar mechanism. Hence, challenge to future strategies should be to fold the disparate and sometimes contradictory data into a harmonized overall picture. [ABSTRACT FROM AUTHOR]

The amino acid sequence of 5-phospho-α-d-ribosyl 1-diphosphate synthase from the thermophileBacillus caldolyticusis 81% identical to the amino acid sequence of 5-phospho-α-d-ribosyl 1-diphosphate synthase from the mesophileBacillus subtilis. Nevertheless the enzyme from the two organisms possesses very different thermal properties. TheB. caldolyticusenzyme has optimal activity at 60–65 °C and a half-life of 26 min at 65 °C, compared to values of 46 °C and 60 s at 65 °C, respectively, for theB. subtilisenzyme. Chemical cross-linking shows that both enzymes are hexamers.Vmax is determined as 440 µmol·min−1·mg protein−1andKm values for ATP and ribose 5-phosphate are determined as 310 and 530 µm, respectively, for theB. caldolyticusenzyme. The enzyme requires 50 mmPi as well as free Mg2+ for maximal activity. Manganese ion substitutes for Mg2+, but only at 30% of the activity obtained with Mg2+. ADP and GDP inhibit theB. caldolyticusenzyme in a cooperative fashion with Hill coefficients of 2.9 for ADP and 2.6 for GDP.Ki values are determined as 113 and 490 µmfor ADP and GDP, respectively. At low concentrations ADP inhibition is linearly competitive with respect to ATP. A predicted structure of theB. caldolyticusenzyme based on homology modelling with the structure ofB. subtilis5-phospho-α-d-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure. [ABSTRACT FROM AUTHOR]

Ferredoxin–NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn2+ (Ki 1.57 µm). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 µm), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions.Escherichia coliFNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes. The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors. Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron-transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected. Zn-ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Ferredoxin reduction was also inhibited by Zn-ferrocyanide in a noncompetitive manner, but the observedKi was about nine times higher than those for the diaphorase reactions. The electron transfer toAnabaenaflavodoxin was not affected by Zn-ferrocyanide. Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect. [ABSTRACT FROM AUTHOR]

The influence of substrates on the interaction of apotransketolase with thiamin diphosphate was investigated in the presence of magnesium ions. It was shown that the donor substrates, but not the acceptor substrates, enhance the affinity of the coenzyme either to only one active center of transketolase or to both active centers, but to different degrees in each, resulting in a negative cooperativity for coenzyme binding. In the absence of donor substrate, negative cooperativity is not observed. The donor substrate did not affect the interaction of the apoenzyme with the inactive coenzyme analogue, N3′-pyridyl-thiamin diphosphate. The influence of the donor substrate on the coenzyme–apotransketolase interaction was predicted as a result of formation of the transketolase reaction intermediate 2-(α,β-dihydroxyethyl)-thiamin diphosphate, which exhibited a higher affinity to the enzyme than thiamin diphosphate. The enhancement of thiamin diphosphate's affinity to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the substrate-affected transketolase regulation at low coenzyme concentrations. [ABSTRACT FROM AUTHOR]

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-α,α-1,1-glucose) and maltose (glucosyl-α1-4-glucose). TreS was purified from the cytosol ofMycobacterium smegmatisto give a single protein band on SDS gels with a molecular mass of≈ 68 kDa. However, active enzyme exhibited a molecular mass of≈ 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, thetreSgene was identified in theM. smegmatisgenome sequence, and was cloned and expressed in active form inEscherichia coli.The recombinant protein was synthesized with a (His)6 tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42–45% of each) was reached during an incubation of about 6 h, whereas at 2 mmmaltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in≥ 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8–10% free glucose. TheKm for maltose was≈ 10 mm, whereas for trehalose it was≈ 90 mm. Whileβ,β-trehalose, isomaltose (α1,6-glucose disaccharide), kojibiose (α1,2) or cellobiose (β1,4) were not substrates for TreS, nigerose (α1,3-glucose disaccharide) andα,β-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer.[3H]Trehalose is converted to[3H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and[14C]maltose produces[14C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as[3H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of[3H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry. [ABSTRACT FROM AUTHOR]

The crystal structure of a heparin cofactor II (HCII)–thrombin Michaelis complex has revealed extensive contacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast, the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy, we demonstrate that at least three different sites (positions 52, 54 and 68) within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule, suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner. Cross-linking of the N terminus to the HCII body does not strongly affect the inhibition ofα-chymotrypsin, indicating that the reactive site loop sequences of the engineered inhibitor variants, required for interaction with one of the HCII target enzymes, are normally accessible. In contrast, intramolecular tethering of the N-terminal extension results in a drastic decrease ofα-thrombin inhibitory activity, both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towardsα-thrombin, suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor andα-thrombin. [ABSTRACT FROM AUTHOR]

d-Ornithine aminomutase fromClostridium sticklandiicomprises two strongly associating subunits, OraS and OraE, with molecular masses of 12 800 and 82 900 Da. Previous studies have shown that inEscherichia colithe recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or adenosylcobalamin (AdoCbl) play important roles in the refolding process. In this study, the DNA fragment containing both genes was cloned into the same expression vector and coexpression of theoraEandoraSgenes was carried out inE. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-β-d-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with aKi of 96 ± 14 µm. Lys629 is responsible for the binding of PLP. The apparentKd for coenzyme B6 binding tod-ornithine aminomutase is 224 ± 41 nmas measured by equilibrium dialysis. The mutant protein, OraSE–K629M, is successfully expressed. It is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding duringin vivotranslation of OraSE–K629M inE. coli,in vitrorefolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important ford-ornithine aminomutase to maintain an active conformation. [ABSTRACT FROM AUTHOR]

In order to study the role of peroxisome proliferator-activated receptorα in mouse intestine, its agonist-induced proteins were identified by peptide mass fingerprinting followed by Northern blot analysis using their cDNAs. One of the most remarkably induced proteins was identified as 17β-hydroxysterol dehydrogenase type 11. Its very rapid induction by various agonists was most efficient in intestine and then in liver. These findings together with recently reported results showing the enzyme family's wide substrate spectrum, including not only glucocorticoids and sex steroids but also bile acids, fatty acids and branched chain amino acids, suggest new roles for both peroxisome proliferator-activated receptorα and 17β-hydroxysterol dehydrogenase type 11 in lipid metabolism and/or detoxification in the intestine. [ABSTRACT FROM AUTHOR]

Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellateEuglena graciliswere purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol ofE. graciliswere cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor-Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase ofE. gracilisshowed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme ofE. gracilisdid not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi. [ABSTRACT FROM AUTHOR]

The peptide sequence DSYG(893–896) of the sheep sodium pumpα1 subunit is highly conserved among all K+-transporting P-type ATPases. To obtain information about its function, single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity, ion recognition, andα/β subunit interactions. Mutants of Ser894 or Tyr895 were all active. Conservative phenylalanine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild-type enzyme. Replacement of the same amino acid by cysteine, however, produced heat-sensitive enzymes, indicating that the aromatic group contributes to the stability of the enzyme. Mutants of the neighbouring Ser894 recognized K+ with altered apparent affinities. Thus, the Ser894→Asp mutant displayed a threefold higher apparent affinity for K+ (EC50 = 1.4 ± 0.06 mm) than the wild-type enzyme (EC50 = 3.8 ± 0.33 mm). In contrast, the mutant Ser894→Ile had an almost sixfold lower apparent affinity for K+ (EC50 = 21.95 ± 1.41 mm). Mutation of Asp893 or Gly896 produced inactive proteins. When an anti-β1 subunit immunoglobulin was used to co-immunoprecipitate theα1 subunit, neither the Gly896→Arg nor the Gly896→Ile mutant could be visualized by subsequent probing with an anti-α1 subunit immunoglobulin. On the other hand, co-immunoprecipitation was obtained with the inactive Asp893→Arg and Asp893→Glu mutants. Thus, it might be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and/or ion translocation. The results obtained here demonstrate the importance of the highly conserved peptide DSYG(893–896) for the function ofα/β heterodimeric P-type ATPases. [ABSTRACT FROM AUTHOR]

S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthesized and shown to be an effective alkylating affinity label for recombinant maize glutathione S-transferase I (GST I). Inactivation of GST I by SDTG at pH 6.5 followed biphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactivation followed by a slower phase, leading to 42 ± 3% residual activity. The rate of inactivation for both phases exhibits nonlinear dependence on SDTG concentration, consistent with the formation of a reversible complex with the enzyme ( Kd 107.9 ± 2.1 µ m for the fast phase, and 224.5 ± 4.2 µ m for the slow phase) before irreversible modification with maximum rate constants of 0.049 ± 0.002 min−1 and 0.0153 ± 0.001 min−1 for the fast and slow phases, respectively. Protection from inactivation was afforded by substrate analogues, demonstrating the specificity of the reaction. When the enzyme was inactivated (42% residual activity), ≈ 1 mol SDTG per mol dimeric enzyme was incorporated. Amino-acid analysis, molecular modelling, and site-directed mutagenesis studies suggested that the modifying residue is Met121, which is located at the end of α-helix H′′′3 and forms part of the xenobiotic-binding site. The results reveal an unexpected structural communication between subunits, which consists of mutually exclusive modification of Met residues across enzyme subunits. Thus, modification of Met121 on one subunit prevents modification of Met121 on the other subunit. This communication is governed by Phe51, which is located at the dimer interface and forms part of the hydrophobic lock-and-key intersubunit motif. The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites. [ABSTRACT FROM AUTHOR]

A detailed comparison between native chlorite dismutase from Ideonella dechloratans, and the recombinant version of the protein produced in Escherichia coli, suggests the presence of a covalent modification in the native enzyme. Although the native and recombinant N- and C-terminal sequences are identical, the enzymes display different electrophoretic mobilities, and produce different peptide maps upon digestion with trypsin and separation of fragments using capillary electrophoresis. Comparison of MALDI mass spectra of tryptic peptides from the native and recombinant enzymes suggests two locations for modification in the native protein. Mass spectrometric analysis of isolated peptides from a tryptic digest of the native enzyme identifies a possible cross-linked dipeptide, suggesting an intrachain cross-link in the parent protein. Spectrophotometric titration of the native enzyme in the denatured state reveals two titrating components absorbing at 295 nm, suggesting the presence of about one tyrosine residue per subunit with an anomalously low p Ka. The EPR spectrum for the recombinant enzyme is different from that of the native enzyme, and contains a substantial contribution of a low-spin species with the characteristics of bis-histidine coordination. These results are discussed in terms of a covalent cross-link between a histidine and a tyrosine sidechain, similar to those found in other heme enzymes operating under highly oxidizing conditions. [ABSTRACT FROM AUTHOR]

The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of human α-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm−1. HHP (270 MPa) shifted the centre of mass by only 370 cm−1. HHP combined with a subdenaturing urea concentration (1.5 m) displaced the centre of mass by ≈ 750 cm−1. The binding of the fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to thrombin was increased by 1.8-, 4.0-, and 2.7-fold after treatment with high urea concentration, HHP or HHP combined with urea, respectively, thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand, treatment of thrombin with urea (but not HHP) combined with dithiothreitol progressively displaced the fluorescent probe, thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein- d-Phe-Pro-Arg-cloromethylketone-α-thrombin: addition of urea up to 2 m gradually decreased the fluorescence emission of the probe to 65% of the initial intensity, whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was enhanced (35%) in 2 m urea and gradually abolished at higher concentrations, while HHP (270 MPa) inhibited the enzyme's catalytic activity by 45% and abolished it when 1.5 m urea was also present. Altogether, analysis of urea and HHP effects on thrombin structure and activity indicates the formation of dissimilar intermediate states during denaturation by these agents. [ABSTRACT FROM AUTHOR]

Differences in the substrate specificity of α-glucosidases should be due to the differences in the substrate binding and the catalytic domains of the enzymes. To elucidate such differences of enzymes hydrolyzing α-1,4- and α-1,6-glucosidic linkages, two α-glucosidases, maltase and isomaltase, from Saccharomyces cerevisiae were cloned and analyzed. The cloned yeast isomaltase and maltase consisted of 589 and 584 amino acid residues, respectively. There was 72.1% sequence identity with 165 amino acid alterations between the two α-glucosidases. These two α-glucosidase genes were subcloned into the pKP1500 expression vector and expressed in Escherichia coli. The purified α-glucosidases showed the same substrate specificities as those of their parent native glucosidases. Chimeric enzymes constructed from isomaltase by exchanging with maltase fragments were characterized by their substrate specificities. When the consensus region II, which is one of the four regions conserved in family 13 (α-amylase family), is replaced with the maltase type, the chimeric enzymes alter to hydrolyze maltose. Three amino acid residues in consensus region II were different in the two α-glucosidases. Thus, we modified Val216, Gly217, and Ser218 of isomaltase to the maltase-type amino acids by site-directed mutagenesis. The Val216 mutant was altered to hydrolyze both maltose and isomaltose but neither the Gly217 nor the Ser218 mutant changed their substrate specificity, indicating that Val216 is an important residue discriminating the α-1,4- and 1,6-glucosidic linkages of substrates. [ABSTRACT FROM AUTHOR]

Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol–disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus ( PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 °C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family. [ABSTRACT FROM AUTHOR]

2-Amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d. In vitro, this compound is nonenzymatically converted to 2,5-pyridinedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-carboxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway, 4-amino-3-hydroxybenzoate 2,3-dioxygenase, to a yellow compound (εmax = 375 nm). The enzyme in the crude extract carrying out the next step was purified to homogeneity. The yellow compound formed from 4-amino-3-hydroxybenzoic acid by this purified enzyme and purified 4-amino-3-hydroxybenzoate 2,3-dioxygenase in a coupled assay was identified as 2-hydroxymuconic 6-semialdehyde by GC-MS analysis. A mechanism for the formation of 2-hydroxymuconic 6-semialdehyde via enzymatic deamination and nonenzymatic decarboxylation is proposed based on results of spectrophotometric analyses. The purified enzyme, designated 2-amino-5-carboxymuconic 6-semialdehyde deaminase, is a new type of deaminase that differs from the 2-aminomuconate deaminases reported previously in that it primarily and specifically attacks 2-amino-5-carboxymuconic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-aminophenol and its derivatives. [ABSTRACT FROM AUTHOR]

Fifty-nine amino acid sequences belonging to family 57 (GH-57) of the glycoside hydrolases were collected using the CAZy server, Pfam database and blast tools. Owing to the sequence heterogeneity of the GH-57 members, sequence alignments were performed using mainly manual methods. Likewise, five conserved regions were identified, which are postulated to be GH-57 consensus motifs. In the 659 amino acid-long 4-α-glucanotransferase from Thermococcus litoralis, these motifs correspond to 13_HQP (region I), 76_GQLEIV (region II), 120_WLTERV (region III), 212_HDDGEKFGVW (region IV), and 350_AQCNDAYWH (region V). The third and fourth conserved regions contain the catalytic nucleophile and the proton donor, respectively. Based on our sequence alignment, residues Glu291 and Asp394 were proposed as the nucleophile and proton donor, respectively, in a GH-57 amylopullulanase from Thermococcus hydrothermalis. To validate this prediction, site-directed mutagenesis was performed. The results of this work reveal that both residues are critical for the pullulanolytic and amylolytic activities of the amylopullulanase. Therefore, these data support the prediction and strongly suggest that the bifunctionality of the amylopullulanase is determined by a single catalytic centre. Despite this positive validation, our alignment also reveals that certain GH-57 members do not possess the Glu and Asp corresponding to the predicted GH-57 catalytic residues. However, the sequences concerned by this anomaly encode putative proteins for which no biochemical or enzymatic data are yet available. Finally, the evolutionary trees generated for GH-57 reveal that the entire family can be divided into several subfamilies that may reflect the different enzyme specificities. [ABSTRACT FROM AUTHOR]

Cellular functions are ultimately linked to metabolic fluxes brought about by thousands of chemical reactions and transport processes. The synthesis of the underlying enzymes and membrane transporters causes the cell a certain ‘effort’ of energy and external resources. Considering that those cells should have had a selection advantage during natural evolution that enabled them to fulfil vital functions (such as growth, defence against toxic compounds, repair of DNA alterations, etc.) with minimal effort, one may postulate the principle of flux minimization, as follows: given the available external substrates and given a set of functionally important ‘target’ fluxes required to accomplish a specific pattern of cellular functions, the stationary metabolic fluxes have to become a minimum. To convert this principle into a mathematical method enabling the prediction of stationary metabolic fluxes, the total flux in the network is measured by a weighted linear combination of all individual fluxes whereby the thermodynamic equilibrium constants are used as weighting factors, i.e. the more the thermodynamic equilibrium lies on the right-hand side of the reaction, the larger the weighting factor for the backward reaction. A linear programming technique is applied to minimize the total flux at fixed values of the target fluxes and under the constraint of flux balance (= steady-state conditions) with respect to all metabolites. The theoretical concept is applied to two metabolic schemes: the energy and redox metabolism of erythrocytes, and the central metabolism of Methylobacterium extorquens AM1 . The flux rates predicted by the flux-minimization method exhibit significant correlations with flux rates obtained by either kinetic modelling or direct experimental determination. Larger deviations occur for segments of the network composed of redundant branches where the flux-minimization method always attributes the total flux to the thermodynamically most favourable branch. Nevertheless, compared with existing methods of structural modelling, the principle of flux minimization appears to be a promising theoretical approach to assess stationary flux rates in metabolic systems in cases where a detailed kinetic model is not yet available. [ABSTRACT FROM AUTHOR]

Jasmonic acid and its methyl ester are ubiquitous plant signalling compounds necessary for the regulation of growth and development, as well as for the response of plants to environmental stress factors. To date, it is not clear whether methyl jasmonate itself acts as a signal or if its conversion to jasmonic acid is mandatory prior to the induction of a defense response. We have cloned a cDNA, encoding a methyl jasmonate-cleaving enzyme, from tomato cell suspension cultures. Sequence analysis revealed significant similarity to plant esterases and to ( S)-hydroxynitrile lyases with an α/β-hydrolase fold structure. The coding sequence was heterologously expressed in Escherichia coli and purified in a catalytically active form. Transcript levels, as well as enzymatic activity, were determined in different tomato tissues. High transcript levels and enzyme activities were found in roots and flowers, while the mRNA level and activity were low in stems and leaves. Moreover, when tested in methyl jasmonate- and elicitor-treated cell suspension cultures, transcript levels were found to decrease, indicating that this particular enzyme might be a regulator of jasmonate signalling. [ABSTRACT FROM AUTHOR]

The properties of the Δ6 desaturase/acetylenase from the moss Ceratodon purpureus and the Δ12 acetylenase from the dicot Crepis alpina were studied by expressing the encoding genes in Arabidopsis thaliana and Saccharomyces cerevisiae. The acetylenase from C. alpinaΔ12 desaturated both oleate and linoleate with about equal efficiency. The desaturation of oleate gave rise to 9( Z),12( E)- and 9( Z),12( Z)-octadecadienoates in a ratio of approximately 3 : 1. Experiments using stereospecifically deuterated oleates showed that the pro-R hydrogen atoms were removed from C-12 and C-13 in the introduction of the 12( Z) double bond, whereas the pro-R and pro-S hydrogen atoms were removed from these carbons during the formation of the 12( E) double bond. The results suggested that the Δ12 acetylenase could accommodate oleate having either a cisoid or transoid conformation of the C12-C13 single bond, and that these conformers served as precursors of the 12( Z) and 12( E) double bonds, respectively. However, only the 9( Z),12( Z)-octadecadienoate isomer could be further desaturated to 9( Z)-octadecen-12-ynoate (crepenynate) by the enzyme. The evolutionarily closely related Δ12 epoxygenase from Crepis palaestina had only weak desaturase activity but could also produce 9( Z),12( E)-octadecadienoate from oleate. The Δ6 acetylenase/desaturase from C. purpureus, on the other hand, produced only the 6( Z) isomers using C16 and C18 acyl groups possessing a Δ9 double bond as substrates. The Δ6 double bond was efficiently further converted to an acetylenic bond by a second round of desaturation but only if the acyl substrate had a Δ12 double bond and that this was in the Z configuration. [ABSTRACT FROM AUTHOR]

In the nonmevalonate pathway of isoprenoid biosynthesis, the conversion of 2 C-methyl- d-erythritol 4-phosphate into its cyclic diphosphate proceeds via nucleotidyl intermediates and is catalyzed by the products of the ispD, ispE and ispF genes. An open reading frame of Campylobacter jejuni with similarity to the ispD and ispF genes of Escherichia coli was cloned into an expression vector directing the formation of a 42 kDa protein in a recombinant E. coli strain. The purified protein was shown to catalyze the transformation of 2 C-methyl- d-erythritol 4-phosphate into 4-diphosphocytidyl-2 C-methyl- d-erythritol and the conversion of 4-diphosphocytidyl-2 C-methyl- d-erythritol 2-phosphate into 2 C-methyl- d-erythritol 2,4-cyclodiphosphate at catalytic rates of 19 µmol·mg−1·min−1 and 7 µmol·mg−1·min−1, respectively. Both enzyme-catalyzed reactions require divalent metal ions. The C. jejuni enzyme does not catalyze the formation of 2 C-methyl- d-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2 C-methyl- d-erythritol, a side reaction catalyzed in vitro by the IspF proteins of E. coli and Plasmodium falciparum. Comparative genomic analysis show that all sequenced α- and ε-proteobacteria have fused ispDF genes. These bifunctional proteins are potential drug targets in several human pathogens (e.g. Helicobacter pylori, C. jejuni and Treponema pallidum). [ABSTRACT FROM AUTHOR]

Subunit b is indispensable for the formation of a functional H+-translocating FO complex both in vivo and in vitro. Whereas the very C-terminus of subunit b interacts with F1 and plays a crucial role in enzyme assembly, the C-terminal region is also considered to be necessary for proper reconstitution of FO into liposomes. Here, we show that a synthetic peptide, residues 1–34 of subunit b ( b1−34) [Dmitriev, O., Jones, P.C., Jiang, W. & Fillingame, R.H. (1999) J. Biol. Chem. 274, 15598–15604], corresponding to the membrane domain of subunit b was sufficient in forming an active FO complex when coreconstituted with purified ac subcomplex. H+ translocation was shown to be sensitive to the specific inhibitor N,N′-dicyclohexylcarbodiimide, and the resulting FO complexes were deficient in binding of isolated F1. This demonstrates that only the membrane part of subunit b is sufficient, as well as necessary, for H+ translocation across the membrane, whereas the binding of F1 to FO is mainly triggered by C-terminal residues beyond Glu34 in subunit b. Comparison of the data with former reconstitution experiments additionally indicated that parts of the hydrophilic portion of the subunit b dimer are not involved in the process of ion translocation itself, but might organize subunits a and c in FO assembly. Furthermore, the data obtained functionally support the monomeric NMR structure of the synthetic b1−34. [ABSTRACT FROM AUTHOR]

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184 997–186 043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 µmol·mg−1·min−1, respectively, as diagnosed by 1H spectroscopy. FMN and divalent cations are required for catalytic activity; the highest rates were found with Ca2+. NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of ( S)-α–hydroxyacid oxidizing enzymes including flavocytochrome b2 and l-lactate dehydrogenase and was shown to catalyse the formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit at a low rate of 1 nmol·mg−1·min−1. Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy. [ABSTRACT FROM AUTHOR]

Enzyme catalysis in low water containing organic solvents is finding an increasing number of applications in diverse areas. This review focuses on some aspects which have not been reviewed elsewhere. Different strategies for obtaining higher activity and stability in such media are described. In this context, the damaging role of lyophilization and the means of overcoming such effects are discussed. Ultrasonication and microwave assistance are two emerging approaches for enhancing reaction rates in low water media. Control of water activity and medium engineering are two crucial approaches in optimization of catalytic behaviour in nonaqueous enzymology. Organometallics and synthesis/modification of polymers are two areas where nonaqueous enzymology can play a greater role in the coming years. The greater understanding of enzyme behaviour in nonaqueous media is expected to lead to larger and even more diverse kinds of applications. [ABSTRACT FROM AUTHOR]

UDP- N-acetylglucosamine enolpyruvyl transferase (MurA), catalyzes the first step in the biosynthesis of peptidoglycan, involving the transfer of the intact enolpyruvyl moiety from phospho enolpyruvate to the 3′-hydroxyl group of UDP- N-acetylglucosamine (UDPNAG). The enzyme is irreversibly inhibited by the antibiotic fosfomycin. The inactivation is caused by alkylation of a highly conserved cysteine residue (C115) that participates in the binding of phospho enolpyruvate. The three-dimensional structure of the enzyme suggests that two residues may play a decisive role in fosfomycin binding: K22 and R120. To investigate the role of these residues, we have generated the K22V, K22E, K22R and R120K single mutant proteins as well as the K22V/R120K and K22V/R120V double mutant proteins. We demonstrated that the K22R mutant protein behaves similarly to wild-type enzyme, whereas the K22E mutant protein failed to form the covalent adduct. On the other hand, the K22V mutant protein requires the presence of UDPNAG for the formation of the adduct indicating that UDPNAG plays a crucial role in the organization of productive interactions in the active site. This model receives strong support from heat capacity changes observed for the K22V/R120K and R120K mutant proteins: in both mutant proteins, the heat capacity changes are markedly reduced indicating that their ability to form a closed protein conformation is impeded due to the R120K exchange. [ABSTRACT FROM AUTHOR]

The prolyl oligopeptidase (POP) family of serine proteases includes prolyl oligopeptidase, dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B. The enzymes of this family specifically hydrolyze oligopeptides with less than 30 amino acids. Many of the POP family enzymes have evoked pharmaceutical interest as they have roles in the regulation of peptide hormones and are involved in a variety of diseases such as dementia, trypanosomiasis and type 2 diabetes. In this study we have clarified the evolutionary relationships of these four POP family enzymes and analyzed POP sequences from different sources. The phylogenetic trees indicate that the four enzymes were present in the last common ancestor of all life forms and that the β-propeller domain has been part of the family for billions of years. There are striking differences in the mutation rates between the enzymes and POP was found to be the most conserved enzyme of this family. However, the localization of this enzyme has changed throughout evolution, as three archaeal POPs seem to be membrane bound and one third of the bacterial as well as two eukaryotic POPs were found to be secreted out of the cell. There are also considerable distinctions between the mutation rates of the different substrate binding subsites of POP. This information may help in the development of species-specific POP inhibitors. [ABSTRACT FROM AUTHOR]

A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27KIP1, in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk . Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100–200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1–10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypothesis whereby KIPase is a currently unidentified caspase-like enzyme which regulates the abundance of p27KIP1 in a proliferation-dependent manner. [ABSTRACT FROM AUTHOR]

Ribonuclease L (RNase L) is a key enzyme in the 2-5A host defense system, and its activity is strictly regulated by an unusual 2′,5′-linked oligoadenylate (2-5A). A bipartite model, in which the N-terminal half of RNase L is responsible for the 2-5A binding and the C-terminal half alone is able to hydrolyse the substrate RNA, has been proposed on the basis of the results of deletion mutant analyses [Dong, B. & Silverman, R.H. (1997) J. Biol. Chem. 272, 22236–22242]. Above all, the region between Glu711 and His720 was revealed to be essential for RNA binding and/or hydrolysis. To dissect the function of the region, we performed scanning mutagenesis over the 10 residues of glutathione S-transferase (GST)-fusion RNase L. Among the single amino acid mutants examined, Y712A and F716A resulted in a significant decrease of RNase activity with a reduced RNA binding acitivity. The losses of the RNase activity were not restored by its conservative mutation, whereas the RNA binding activity was enhanced in the case of Y712F. These results indicate that both Tyr712 and Phe716 provide the enzyme with a RNA binding activity and catalytic environment. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of d-glucose-6-phosphate to d-fructose-6-phosphate in glycolysis and gluconeogenesis, and in the recycling of hexose-6-phosphate in the pentose phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order Kinetoplastida are important human parasites responsible for African sleeping sickness, Chagas' disease and leishmaniases, respectively. In these parasites, glycolysis is an important (and in some cases the only) metabolic pathway for ATP supply. The first seven of the 10 enzymes that participate in glycolysis, as well as an important fraction of the enzymes of the pentose phosphate pathway, are compartmentalized in peroxisome-like organelles called glycosomes. The dependence of the parasites on glycolysis, the importance of the pentose phosphate pathway in defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes contained in the glycosome as potential targets for drug design. The present report describes the first crystallographic structure of a parasite ( Leishmania mexicana) glucose-6-phosphate isomerase. A comparison of the atomic structure of L. mexicana, human and other mammalian PGIs, which highlights unique features of the parasite's enzyme, is presented. [ABSTRACT FROM AUTHOR]

Little is known about the functions of DNA polymerase λ (Pol λ) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol λ may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol λ in a higher plant, rice ( Oryza sativa L. cv. Nipponbare) ( OsPol λ). OsPol λ had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol λ. Northern blot analysis showed that the level of OsPol λ expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol λ is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol β and terminal deoxyribonucleotidyl transferase. [ABSTRACT FROM AUTHOR]

The pyrG gene from Lactococcus lactis encodes CTP synthase (EC 6.4.3.2), an enzyme converting UTP to CTP. A series of strains were constructed with different levels of pyrG expression by insertion of synthetic constitutive promoters with different strengths in front of pyrG. These strains expressed pyrG levels in a range from 3 to 665% relative to the wild-type expression level. Decreasing the level of CTP synthase to 43% had no effect on the growth rate, showing that the capacity of CTP synthase in the cell is in excess in a wild-type strain. We then studied how pyrG expression affected the intracellular pool sizes of nucleotides and the correlation between pyrG expression and nucleotide pool sizes was quantified using metabolic control analysis in terms of inherent control coefficients. At the wild-type expression level, CTP synthase had full control of the CTP concentration with a concentration control coefficient close to one and a negative concentration control coefficient of −0.28 for the UTP concentration. Additionally, a concentration control coefficient of 0.49 was calculated for the dCTP concentration. Implications for the homeostasis of nucleotide pools are discussed. [ABSTRACT FROM AUTHOR]

Numerous transmembrane proteins, including the blood pressure regulating angiotensin converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein (APP), are proteolytically shed from the plasma membrane by metalloproteases. We have used an antisense oligonucleotide (ASO) approach to delineate the role of ADAM10 and tumour necrosis factor-α converting enzyme (TACE; ADAM17) in the ectodomain shedding of ACE and APP from human SH-SY5Y cells. Although the ADAM10 ASO and TACE ASO significantly reduced (> 81%) their respective mRNA levels and reduced the α-secretase shedding of APP by 60% and 30%, respectively, neither ASO reduced the shedding of ACE. The mercurial compound 4-aminophenylmercuric acetate (APMA) stimulated the shedding of ACE but not of APP. The APMA-stimulated secretase cleaved ACE at the same Arg-Ser bond in the juxtamembrane stalk as the constitutive secretase but was more sensitive to inhibition by a hydroxamate-based compound. The APMA-activated shedding of ACE was not reduced by the ADAM10 or TACE ASOs. These results indicate that neither ADAM10 nor TACE are involved in the shedding of ACE and that APMA, which activates a distinct ACE secretase, is the first pharmacological agent to distinguish between the shedding of ACE and APP. [ABSTRACT FROM AUTHOR]

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC50 of 19.8 µ m. Interestingly, its cleavage product, IPEP exhibits an IC50 of 11.5 µ m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 µ m IPEP and 35% inhibition with LORF (25 m m). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates. [ABSTRACT FROM AUTHOR]

The Baeyer–Villiger monooxygenase (BVMO), 4-hydroxyacetophenone monooxygenase (HAPMO), uses NADPH and O2 to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conserved basic residues, which could account for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. For Arg440 no involvement in coenzyme recognition could be shown as mutant R440A was totally inactive. Although this mutant could still be fully reduced by NADPH, no oxygenation occurred, indicating that this residue is crucial for completing the catalytic cycle of HAPMO. Characterization of several Arg339 and Lys439 mutants revealed that these residues are indeed both involved in coenzyme recognition. Mutant R339A showed a largely decreased affinity for NADPH, as judged from kinetic analysis and binding experiments. Replacing Arg339 also resulted in a decreased catalytic efficiency with NADH. Mutant K439A displayed a 100-fold decrease in catalytic efficiency with NADPH, mainly caused by an increased Km. However, the efficiency with NADH increased fourfold. Saturation mutagenesis at position 439 showed that the presence of an asparagine or a phenylalanine improves the catalytic efficiency with NADH by a factor of 6 to 7. All Lys439 mutants displayed a lower affinity for AADP+, confirming a role of the lysine in recognizing the 2′-phosphate of NADPH. The results obtained could be extrapolated to the sequence-related cyclohexanone monooxygenase. Replacing Lys326 in this BVMO, which is analogous to Lys439 in HAPMO, again changed the coenzyme specificity towards NADH. These results indicate that the strict NADPH dependency of this class of monooxygenases is based upon recognition of the coenzyme by several basic residues. [ABSTRACT FROM AUTHOR]

Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana, new questions concerning novel structure–function relationships among plant aspartic proteinases are now starting to be addressed. [ABSTRACT FROM AUTHOR]

Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[14C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d, l- threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [14C]palmitic acid and [14C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry. When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy. [ABSTRACT FROM AUTHOR]

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (β2), the recombinant enzyme was a hetero-octameric (β4γ4), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U·mg−1. Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system. [ABSTRACT FROM AUTHOR]

Information on the regulation and structure–function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer–tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern. [ABSTRACT FROM AUTHOR]

Kinetic experiments with a substrate series of phenylacetyl-arylamides reveal that at least one polar group in the amine moiety is required for the proper orientation of the substrate in the large nucleophile-binding subsite of penicillin acylase of Escherichia coli. Quantum mechanical molecular modelling of enzyme–substrate interactions in the enzyme active site shows that in the case of substrates lacking local symmetry, the productive binding implies two nonsymmetrical arrangements with respect to the two positively charged guanidinium residues of ArgA145 and ArgB263. This indicates a crucial role of the specified arginine pair in the substrate- and stereoselectivity of penicillin acylase. [ABSTRACT FROM AUTHOR]

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3′ to 5′ or 5′ to 3′ direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables. [ABSTRACT FROM AUTHOR]

DNA helicases are molecular motor enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases. [ABSTRACT FROM AUTHOR]

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes L -arginine to L -ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity. A translationally fused His6­RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase-containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His6­RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His6­RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism. [ABSTRACT FROM AUTHOR]

Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC [Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation. ActivatedHaCA42carboxypeptidasehydrolysedasynthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects. [ABSTRACT FROM AUTHOR]

Studies in knockout mice support the involvement of alcohol dehydrogenases ADH 1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11-cisretinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all- trans-, 7-cis-, 9-cis-, 1 1-cis- and 13-cis-isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 1 3-cis isomers which are not used by ADH 1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 1 1-cis-retinoids efficiently. ADH4 shows much higher kcat/Km values for 1 1-cis-retinol oxidation than for 1 1-cis-retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 1 1-cis- retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis. [ABSTRACT FROM AUTHOR]

The efficient engineering of enzymes with novel activities remains an ongoing challenge. Towards this end, genetic selection techniques provide a method for finding rare solutions to catalytic problems that requires only a limited foreknowledge of structure-function relationships. We have used genetic selections to extensively probe the structure and mechanism of chorismate mutases. The insights gained from these investigations will aid future enzyme design efforts. [ABSTRACT FROM AUTHOR]