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Pullulanases are important starch-debranching enzymes that mainly hydrolyze the α-1,6-glycosidic linkages in pullulan, starch, and oligosaccharides. Nevertheless, their practical applications are constrained because of their poor activity and low thermostability. Moreover, the trade-off between activity and thermostability makes it challenging to simultaneously improve them. In this study, an engineered pullulanase was developed through reshaping the active-site tunnel and engineering the surface lysine residues using the pullulanase from Pyrococcus yayanosii CH1 (Pul PY2 ). The specific activity of the engineered pullulanase was increased 3.1-fold, and thermostability was enhanced 1.8-fold. Moreover, the engineered pullulanase exhibited 11.4-fold improvement in catalytic efficiency (k cat /K m ). Molecular dynamics simulations demonstrated an anti-correlated movement around the entrance of active-site tunnel and stronger interactions between the surface residues in the engineered pullulanase, which would be beneficial to the activity and thermostability improvement, respectively. The strategies used in this study and dynamic evidence for insight into enzyme performance improvement may provide guidance for the activity and thermostability engineering of other enzymes., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heat shock proteins (HSPs) are known to confer protection to the stressed cells by rescuing vital host cell proteins. In the present study we have demonstrated that heterologous expression of N-terminal domain of hyperthermophilic L-asparaginase (NPfA) confers thermotolerance to E. coli. The recombinant expression of NPfA enabled E. coli to demonstrate typical growth behavior at 52°C and survive a thermal shock up to 62°C, both being the highest reported temperatures for growth and heat shock survival. To understand the basis of protection proteome analysis of these cells was carried out which showed that NPfA guards a battery of proteins, especially related to gene regulations and repair, providing definite survival advantage to the stressed cells. Thus NPfA a non-canonical, non-natural chaperone has been shown to render E. coli cells with selective growth advantage under extremes of conditions. We propose that such modified, heat stabilized hosts could be utilized in developing heat-induced expression systems as well for the recombinant expression of thermophilic proteins., (Copyright © 2017. Published by Elsevier B.V.)

DNA polymerases are useful tools in various biochemical experiments. We have focused on the DNA polymerases involved in DNA replication including the unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px). Many reports have described the different combinations between unnatural base pairs and DNA polymerases. As an example, for the replication of the Ds-Px pair, Deep Vent DNA polymerase exhibits high efficiency and fidelity, but Taq DNA polymerase shows much lower efficiency and fidelity. In the present study, we determined the crystal structure of Deep Vent DNA polymerase in the apo form at 2.5 Å resolution. Using this structure, we constructed structural models of Deep Vent DNA polymerase complexes with DNA containing an unnatural or natural base in the replication position. The models revealed that the unnatural Ds base in the template-strand DNA clashes with the side-chain oxygen of Thr664 in Taq DNA polymerase, but not in Deep Vent DNA polymerase., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS (13)C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN-GlcNAc, (GlcN)2-GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosanase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Triosephophate isomerase (TIM) is a dimeric enzyme in eucarya, bacteria and mesophilic archaea. In hyperthermophilic archaea, however, TIM exists as a tetramer composed of monomers that are about 10% shorter than other eucaryal and bacterial TIM monomers. We report here the crystal structure of TIM from Thermoproteus tenax, a hyperthermophilic archaeon that has an optimum growth temperature of 86 degrees C. The structure was determined from both a hexagonal and an orthorhombic crystal form to resolutions of 2.5A and 2.3A, and refined to R-factors of 19.7% and 21.5%, respectively. In both crystal forms, T.tenax TIM exists as a tetramer of the familiar (betaalpha)(8)-barrel. In solution, however, and unlike other hyperthermophilic TIMs, the T.tenax enzyme exhibits an equilibrium between inactive dimers and active tetramers, which is shifted to the tetramer state through a specific interaction with glycerol-1-phosphate dehydrogenase of T.tenax. This observation is interpreted in physiological terms as a need to reduce the build-up of thermolabile metabolic intermediates that would be susceptible to destruction by heat. A detailed structural comparison with TIMs from organisms with growth optima ranging from 15 degrees C to 100 degrees C emphasizes the importance in hyperthermophilic proteins of the specific location of ionic interactions for thermal stability rather than their numbers, and shows a clear correlation between the reduction of heat-labile, surface-exposed Asn and Gln residues with thermoadaptation. The comparison confirms the increase in charged surface-exposed residues at the expense of polar residues.

Alkaline phosphatases (APs), E.C. 3.1.3.1, are non-specific phosphomonoesterases optimally active under alkaline conditions. They are classically known to be homodimeric metalloenzymes. This quaternary structure has been considered necessary for activity, although the relationship between quaternary structure and activity is not well understood. Recombinant Pyrococcus abyssi AP was previously isolated and characterized, appearing to have two active quaternary structures on native polyacrylamide gel electrophoresis: a monomer and a homodimer. The purpose of the present work was to determine the actual quaternary structure of P. abyssi AP in solution, by isolating each of the two quaternary forms and establishing the parameters governing the assembly and dissociation of the dimer. pH appeared to be an important parameter: in acidic media, the monomer/dimer ratio shifted towards monomer. Buffer composition also affected the quaternary structure: at the same pH, in potassium phosphate buffer, the two quaternary structures were observed, whereas in tris(hydroxymethyl)aminomethane hydrochloride buffer, only the dimer was observed. Metals bound to the enzyme were found to be involved in the stability of the quaternary structure. Indeed, the P. abyssi AP obtained upon removal of the metals was monomeric. Reactivation of the latter was achieved with variable efficiency. From these experiments, no active monomer could be isolated, leading the conclusion that the active form of P. abyssi AP is the homodimer.

Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5(') end of matured tRNA molecules. A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively. These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies. The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P. horikoshii. All four proteins exhibited the binding activity to the RNase P RNA. In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P. horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity. However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity. The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P.

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.

The Pyrococcus abyssi aspartate transcarbamylase (ATCase) shows a high degree of structural conservation with respect to the well-studied mesophilic Escherichia coli ATCase, including the association of catalytic and regulatory subunits. The adaptation of its catalytic function to high temperature was investigated, using enzyme purified from recombinant E.coli cells. At 90 degrees C, the activity of the trimeric catalytic subunit was shown to be intrinsically thermostable. Significant extrinsic stabilization by phosphate, a product of the reaction, was observed when the temperature was raised to 98 degrees C. Comparison with the holoenzyme showed that association with regulatory subunits further increases thermostability. To provide further insight into the mechanisms of its adaptation to high temperature, the crystal structure of the catalytic subunit liganded with the analogue N-phosphonacetyl-L-aspartate (PALA) was solved to 1.8A resolution and compared to that of the PALA-liganded catalytic subunit from E.coli. Interactions with PALA are strictly conserved. This, together with the similar activation energies calculated for the two proteins, suggests that the reaction mechanism of the P.abyssi catalytic subunit is similar to that of the E.coli subunit. Several structural elements potentially contributing to thermostability were identified: (i) a marked decrease in the number of thermolabile residues; (ii) an increased number of charged residues and a concomitant increase of salt links at the interface between the monomers, as well as the formation of an ion-pair network at the protein surface; (iii) the shortening of three loops and the shortening of the N and C termini. Other known thermostabilizing devices such as increased packing density or reduction of cavity volumes do not appear to contribute to the high thermostability of the P.abyssi enzyme.

There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase., (Copyright 2002 Elsevier Science Ltd.)

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT., ((c) 2002 Elsevier Science Ltd.)

New hyperthermostable aminopeptidase from the hyperthermophilic archaeon Pyrococcus horikoshii has acylamino acid releasing (deblocking) activity for acyl (blocked) peptides. Such an enzyme can be used for N-terminal sequencing of acyl peptides. To clarify the active site of the deblocking aminopeptidase, we prepared three mutants in which one of the three possible active site amino acid residues (Asp or Glu) was replaced with their amide derivatives. Activity and cobalt ion dependence of these mutants were examined and compared with those of the native enzyme. The results suggest that all the three possible residues (Asp173, Glu205, and Glu206) participate in the catalytic activity through binding with the cobalt ion.

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base., (Copyright 2001 Academic Press.)

Comparative structural studies on proteins derived from organisms with growth optima ranging from 15 to 100 degrees C are beginning to shed light on the mechanisms of protein thermoadaptation. One means of sustaining hyperthermostability is for proteins to exist in higher oligomeric forms than their mesophilic homologues. Triosephosphate isomerase (TIM) is one of the most studied enzymes, whose fold represents one of nature's most common protein architectures. Most TIMs are dimers of approximately 250 amino acid residues per monomer. Here, we report the 2.7 A resolution crystal structure of the extremely thermostable TIM from Pyrococcus woesei, a hyperthermophilic archaeon growing optimally at 100 degrees C, representing the first archaeal TIM structure. P. woesei TIM exists as a tetramer comprising monomers of only 228 amino acid residues. Structural comparisons with other less thermostable TIMs show that although the central beta-barrel is largely conserved, severe pruning of several helices and truncation of some loops give rise to a much more compact monomer in the small hyperthermophilic TIM. The classical TIM dimer formation is conserved in P. woesei TIM. The extreme thermostability of PwTIM appears to be achieved by the creation of a compact tetramer where two classical TIM dimers interact via an extensive hydrophobic interface. The tetramer is formed through largely hydrophobic interactions between some of the pruned helical regions. The equivalent helical regions in less thermostable dimeric TIMs represent regions of high average temperature factor. The PwTIM seems to have removed these regions of potential instability in the formation of the tetramer. This study of PwTIM provides further support for the role of higher oligomerisation states in extreme thermal stabilisation., (Copyright 2001 Academic Press.)

The crystal structure of family B DNA polymerase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) was determined. KOD DNA polymerase exhibits the highest known extension rate, processivity and fidelity. We carried out the structural analysis of KOD DNA polymerase in order to clarify the mechanisms of those enzymatic features. Structural comparison of DNA polymerases from hyperthermophilic archaea highlighted the conformational difference in Thumb domains. The Thumb domain of KOD DNA polymerase shows an "opened" conformation. The fingers subdomain possessed many basic residues at the side of the polymerase active site. The residues are considered to be accessible to the incoming dNTP by electrostatic interaction. A beta-hairpin motif (residues 242-249) extends from the Exonuclease (Exo) domain as seen in the editing complex of the RB69 DNA polymerase from bacteriophage RB69. Many arginine residues are located at the forked-point (the junction of the template-binding and editing clefts) of KOD DNA polymerase, suggesting that the basic environment is suitable for partitioning of the primer and template DNA duplex and for stabilizing the partially melted DNA structure in the high-temperature environments. The stabilization of the melted DNA structure at the forked-point may be correlated with the high PCR performance of KOD DNA polymerase, which is due to low error rate, high elongation rate and processivity.

We previously reported the presence of a highly active, carboxylase-specific ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. In this study, structural analysis of Pk -Rubisco has been performed. Phylogenetic analysis of Rubiscos indicated that archaeal Rubiscos, including Pk -Rubisco, were distinct from previously reported type I and type II enzymes in terms of primary structure. In order to investigate the existence of small subunits in native Pk -Rubisco, immunoprecipitation and native-PAGE experiments were performed. No specific protein other than the expected large subunit of Pk -Rubisco was detected when the cell-free extracts of P. kodakaraensis KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme. Furthermore, native and recombinant Pk -Rubiscos exhibited identical mobilities on native-PAGE. These results indicated that native Pk -Rubisco consisted solely of large subunits. Electron micrographs of purified recombinant Pk -Rubisco displayed pentagonal ring-like assemblies of the molecules. Crystals of Pk -Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 A resolution. The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly to each other. These results, along with the molecular mass of Pk -Rubisco estimated from gel filtration, strongly suggest that Pk -Rubisco is a decamer composed only of large subunits, with pentagonal ring-like structure. This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos., (Copyright 1999 Academic Press.)

The crystal structure of O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) of hyperthermophilic archaeon Pyrococcuskodakaraensis strain KOD1 (Pk -MGMT) was determined by single isomorphous replacement method with anomalous scattering (SIRAS) at 1.8 A resolution. The archaeal protein is extremely thermostable and repairs alkylated DNA by suicidal alkyl transfer from guanine O6 to its own cysteine residue. Archaea constitute the third primary kingdom of living organisms, sharing characteristics with procaryotic and eucaryotic cells. They live in various extreme environments and are thought to include the most ancient organisms on the earth. Structural studies on hyperthermophilic archaeal proteins reveal the structural features essential for stability under the extreme environments in which these organisms live, and will provide the structural basis required for stabilizing various mesophilic proteins for industrial applications. Here, we report the crystal structure of Pk-MGMT and structural comparison of Pk-MGMT and methyltransferase homologue from Escherichia coli (AdaC, C-terminal fragment of Ada protein). Analyses of solvent-accessible surface area (SASA) reveals a large discrepancy between Pk-MGMT and AdaC with respect to the property of the ASA. In the Pk-MGMT structure, the intra-helix ion-pairs contribute to reinforce stability of alpha-helices. The inter-helix ion-pairs exist in the interior of Pk-MGMT and stabilize internal packing of tertiary structure. Furthermore, structural features of helix cappings, intra and inter-helix ion-pairs are found around the active-site structure in Pk-MGMT., (Copyright 1999 Academic Press.)

Intersubunit ion pairs are considered to be involved for maintaining a stable structure of the glutamate dehydrogenase (GDH) from hyperthermophiles. In order to demonstrate an effect of intersubunit ion pairs on the structural stability, two kinds of mutation (T138E, Thr at position 138 was replaced by Glu; E158Q, Glu at position 158 was replaced by Gln) which add and remove ion pairs, respectively, were introduced into Pk-gdhA gene encoding GDH from Pyrococcus kodakaraensis KOD1. Addition of one ion pair (Pk-GDHA-T138E) increased the optimum temperature and thermostability. In contrast, Pk-GDH-E158Q showed lower optimum temperature and less thermostability than wild type GDH. Structure analysis of GDHs was performed by circular dichroism (CD) and indicated that all recombinant enzymes (Pk-GDH, Pk-GDH-T138E, Pk-GDH-E158Q) possess different structures from that of natural GDH. Upon heat treatment (60 degrees C, 2 h), the structures of Pk-GDH and Pk-GDH-T138E were converted to another form close to the natural structure. However, no structural conversion by heat treatment was observed in Pk-GDH-E158Q. These results indicate that intersubunit ion pairs play an important role in forming thermostable structure of Pk-GDH.

For larger proteins, efficient deblocking prior to Edman sequencing is especially important to obtain quality, extended sequencing data which is limited by the stepwise accumulation of background from the random acid hydrolysis of the protein. Therefore, any portion that remains blocked contributes to the undesirable background. We report an optimized procedure for the removal of pyroglutamate (pGlu) by pyroglutamate aminopeptidase (PGAP) and demonstrate its use for the quantitative deblocking of several humanized recombinant antibodies (rIgGs). The rIgGs with blocked heavy chain provided an advantageous system in which removal of pGlu from the heavy chain was determined as a ratio of the deblocked heavy chain to the light chain in the first cycle of sequencing; i.e., the light chain was used as an internal standard. The reaction temperature, reaction time, enzyme-to-substrate ratio, denaturation, and reduction/carboxymethylation prior to digestion, and different commercial enzymes were evaluated. The optimized procedure involves reduction/carboxymethylation in guanidine buffer, buffer exchange by gel-permeation chromatography, and overnight PGAP digestion at 37 degrees C. Five different rIgGs, including one with blocked heavy and light chains, were deblocked in nearly quantitative yields using this procedure., (Copyright 1998 Academic Press.)

Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus sp. KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in Escherichia coli. Determination of the molecular weight of these enzymes by SDS-PAGE and gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified as both monomeric and hexameric forms. Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active. Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and that circular dichroism spectra of these enzymes are distinctly different from each other. These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II). Upon heat treatment (80 degrees C, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6-fold. Likewise, upon heat treatment (70 degrees C for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated with the hexameric form. These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH.