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We describe a sensitive metabolite array for genome sequence-independent functional analysis of metabolic phenotypes and networks, the reactomes, of cell populations and communities. The array includes 1676 dye-linked substrate compounds collectively representing central metabolic pathways of all forms of life. Application of cell extracts to the array leads to specific binding of enzymes to cognate substrates, transformation to products, and concomitant activation of the dye signals. Proof of principle was shown by reconstruction of the metabolic maps of model bacteria. Utility of the array for unsequenced organisms was demonstrated by reconstruction of the global metabolisms of three microbial communities derived from acidic volcanic pool, deep-sea brine lake, and hydrocarbon-polluted seawater. Enzymes of interest are captured on nanoparticles coated with cognate metabolites, sequenced, and their functions unequivocally established.

A metalloprotease induced by chitin in a new chitinolytic bacterium Serratia sp. Strain KCK was purified and characterized. Compared with other Serratia enzymes, it exhibited a rather broad pH activity range (pH 5.0–8.0), and thermostability. The cognate ORF, mpr, was cloned and expressed. Its deduced amino acid sequence showed high similarity to those of bacterial zinc-binding metalloproteases and a well-conserved serralysin family motif. Pretreatment of chitin with the Mpr protein promoted chitin degradation by chitinase A, which suggests that Mpr participates in, and facilitates, chitin degradation by this microorganism. [ABSTRACT FROM AUTHOR]

The novel chitinolytic bacterium Serratia sp. KCK, which was isolated from kimchi juice, produced chitinase A. The gene coding for the chitinolytic enzyme was cloned on the basis of sequencing of internal peptides, homology search, and design of degenerated primers. The cloned open reading frame of chiA encodes for deduced polypeptide of 563 amino acid residues with a calculated molecular mass of 61 kDa and appears to correspond to a molecular mass of about 57 kDa, which excluded the signal sequence. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified as family 18 of glycosyl hydrolases. The chitinase A is an exochitinase and exhibits a greater pH range (5.0–10.0), thermostability with a temperature optimum of 40°C, and substrate range other than Serratia chitinases thus far described. These results suggested that Serratia sp. KCK chitinase A can be used for biotechnological applications with good potential. [ABSTRACT FROM AUTHOR]

A wide range of microorganisms, the so-called acidophiles, inhabit acidic environments and grow optimally at pH values between 0 and 3. The intracellular pH of these organisms is, however, close to neutrality or slightly acidic. It is to be expected that enzymatic activities dedicated to extracellular functions would be adapted to the prevailing low pH of the environment (0–3), whereas intracellular enzymes would be optimally active at the near-neutral pH of the cytoplasm (4.6–7.0). The genes of several intracellular or cell-bound enzymes, a carboxylesterase and three α-glucosidases, from Ferroplasma acidiphilum, a cell wall-lacking acidophilic archaeon with a growth optimum at pH 1.7, were cloned and expressed in Escherichia coli, and their products purified and characterized. The Ferroplasmaα-glucosidases exhibited no sequence similarity to known glycosyl hydrolases. All enzymes functioned and were stable in vitro in the pH range 1.7–4.0, and had pH optima much lower than the mean intracellular pH of 5.6. This ‘pH optimum anomaly’ suggests the existence of yet-undetected cellular compartmentalization providing cytoplasmic pH patchiness and low pH environments for the enzymes we have analysed. [ABSTRACT FROM AUTHOR]

An enzyme of Alcaligenes eutrophus JMP 134 which catalyzes dechlorination of (4R. 5R)- and (4R, 5S)-5-chloro-3-methyl- and (4R, 5S)-5-chloromuconolactone to principally 3-methyl-trans-, 3-methyl-cis-dienelactone and cis-dienelactone, respectively, was purified to homogeneity. The enzyme was identified as muconolactone isomerase on the basis of its high activity with muconolactone and on its high degree of sequence similarity with previously described muconolactone isomerases. Molecular mass determinations of the highly hydrophobic and heat-resistant enzyme indicated a decameric structure involving a single 10.100 kDa subunit similar to that of muconolactone isomerase of Pseudomonas patida. Kinetic analysis showed cooperative effects between the subunits during conversion of (4R. 5S-5-chloro-3-methylmuconolactone. (4R, 5S)-5-chloromuconolactone was the preferred substrate, over the natural substrate (4S)-muconolactone. The (4S. 5S)-structure was found to be an inhibitor of (4R. 5R)-5-chloro-3 methylmuconolactone transformation. Methylsubstitution of the substrate results in a higher affinity for the enzyme, but a drastically lower velocity, resulting in a lower specificity constant. [ABSTRACT FROM AUTHOR]

A soluble cytochrome P450 whose synthesis is induced by and that binds 2-ethoxyphenol was purified to apparent homogeneity from Rhodococcus rhodochrous strain 116. The enzyme had a subunit molecular mass of 44.5 kDa as determined by SDS/PAGE and a pl of 5.2. The electronic absorption spectrum indicates that the native cytochrome in the absence of substrate is predominantly in the low-spin state (13% high-spin state in 50 mM Mops. pH 7.0, 25°C). 2-Methoxyphenol binds to the cytochrome with a macroscopic dissociation constant of 0.53 ±1- 0.03 μM (50 mM Mops. pH 7.0, 25°C) and induces a 99.7% translation of the heme iron to the pentacoordinate high-spin form. Using a reconstituted in-vitro activity assay. it was demonstrated that P450RRl catalyzed the O-dealkylation of 2-ethoxyphenol and 2-methoxyphenol to produce catechol. The cytochrome binds other ortho-substituted phenols, including 2-ethoxyphenol, 2-methylphenol (o-cresol) and 2-chlorophenol. The affinity of P450RRI for these compounds is lower than that of 2-methoxyphenol and they are less effective than 2-methoxyphenol at inducing a transition in the heme iron to the high-spin state. Para-substituted and meta-substituted ether phenols did not induce a spin transition. [ABSTRACT FROM AUTHOR]

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coil revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) ⪢ phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μ and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases. [ABSTRACT FROM AUTHOR]

Summary: We created a metagenome expression library from the brine:seawater interface of the Urania hypersaline basin, screened it for esterases, and characterized five of these. Two had no significant sequence homology to known esterases, hydrolyzed both carboxylesters and thioesters, and exhibited unusual, habitat-specific characteristics (preference for high hydrostatic pressure and salinity). One has an unusual structural signature incorporating three catalytic active centers mediating distinct hydrolytic activities and an adaptive tertiary-quaternary structure that alters between three molecular states, according to the prevailing physicochemical conditions. Some of the esterases have high activities, specificities, enantioselectivities, and exceptional stability in polar solvents, and they are therefore potentially useful for industrial biotransformations. One possesses the highest enantioselectivity toward an ester of the important chiral synthon solketal (E: 126[S]; 98%ee). [Copyright &y& Elsevier]

Alcanivorax borkumensis is a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This "hydrocarbonoclastic" bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-known alkB1 gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating β-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded by A. borkumensis and its ecological success in oil-contaminated marine habitats. [ABSTRACT FROM AUTHOR]