• BMB Reports
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• v.42 no.1
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• pp.47-52
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• 2009

Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent $K_m$ of 3 mM for L-alanine, and a $V_{max}$ of $295\;{\mu}moles/min/mg$, with the optimum activity occurring at $37^{\circ}C$ and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a $K_i$of $160\;{\mu}M$ and 30 mM, respectively.

• Molecules and Cells
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• v.19 no.1
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• pp.97-103
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• 2005

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the $K_m$ of the enzyme for NADH and ${\alpha}-ketoglutarate$ increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency ($k_{cat}/K_m$) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in $k_{cat}$ values. There was a linear relationship between incorporation of [$^{14}C$]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [$^{14}C$] incorporated per mol of monomer for wild type hGDHs. No incorporation of [$^{14}C$]p-chloromercuribenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.

• BMB Reports
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• v.35 no.5
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• pp.498-507
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• 2002

A gene that encodes a thermostable protease, coined thermicin, has been isolated from Thermoanaerobacter yonseiensis that is expressed and characterized in E. coli.. In order to elucidate the molecular characteristics on thermostability of the enzyme, molecular modeling and mutagenesis technology were applied. In the modeling structure, the structural core, including the active site, was well conserved; whereas, the two loop regions were unique when compared to thermitase. The mutant enzyme with the small loop deleted (D190-I196), based on modeling structural information, showed identical enzyme activity. However, when the large loop was deleted (P233-P244), a little lower $K_m$ and even a lower kcat was found. This indicates that the large loop could influence catalytic activity. However, the unfolding temperature ($T_m$), which was determined by a differential-scanning calorimetry for the mutant enzyme deleted the small loop, was $96^{\circ}C$. This is $14^{\circ}C$ lower than that for the parent thermicin. These results suggest that the small loop may play a role in maintaining the proper folding of the enzyme at high temperatures, whereas the large loop might be related to catalysis.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.242-248
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• 2002

Previously, it was reported that the nonhomologous C-terminal regions of the D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure of the enzyme [3]. In an effort to further confirm the above observation, the C-terminal region-inserted enzyme was constructed by attaching a peptide (22 residues) at the C-terminal of the D-hydantoinase from Bacillus thermocatenulatus GH2, and its structural and biochemical properties were compared with both the wild-type and C-terminal region-truncated enzymes. As a result, native tetrameric D-hydantoinase was dimerized as the truncated enzyme, and the inserted mutant with a new sequence was expressed as a catalytically active form in E. coli. Expression level of the inserted and truncated enzymes were found to be significantly increased compared to the level of the wild-type enzyme, and this appears to be due to the reduced toxic effect of the mutant enzymes on host cells. Dimerized enzymes exhibited increased thermo- and pH stabilities considerably when compared with the corresponding wild-type enzyme. Comparison of the substrate specificity between the mutant and wild-type enzymes suggests that the substrate specificity of the D-hydantoinase is closely linked with the oligomeric structure.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.666-674
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• 2009

An electrochemical bioreactor (ECB) composed of a cathode compartment and an air anode was used in this study to characterize the ethanol fermentation of Zymomonas mobilis. The cathode and air anode were constructed of modified graphite felt with neutral red (NR) and a modified porous carbon plate with cellulose acetate and porous ceramic membrane, respectively. The air anode operates as a catalyst to generate protons and electrons from water. The growth and ethanol production of Z. mobilis were 50% higher in the ECB than were observed under anoxic nitrogen conditions. Ethanol production by growing cells and the crude enzyme of Z. mobilis were significantly lower under aerobic conditions than under other conditions. The growing cells and crude enzyme of Z. mobilis did not catalyze ethanol production from pyruvate and acetaldehyde. The membrane fraction of crude enzyme catalyzed ethanol production from glucose, but the soluble fraction did not. NADH was oxidized to $NAD^+$in association with $H_2O_2$reduction, via the catalysis of crude enzyme. Our results suggested that NADH/$NAD^+$balance may be a critical factor for ethanol production from glucose in the metabolism of Z. mobilis, and that the metabolic activity of both growing cells and crude enzyme for ethanol fermentation may be induced in the presence of glucose.

• BMB Reports
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• v.49 no.6
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• pp.349-354
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• 2016

The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues.

• Journal of Microbiology
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• v.44 no.1
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• pp.50-53
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• 2006

Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase(ValDH) were highly conserved in the corresponding region of $NAD(P)^+$-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.

• Journal of the Korean Magnetic Resonance Society
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• v.21 no.3
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• pp.85-89
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• 2017

Pyrazinamidase (PncA) from Mycobacterium tuberculosis is the hydrolytic enzyme (hydrolase) that can hydrolyze substrate PZA to active form pyrazoic acid (POA). To investigate hydrolytic reaction of M. tuberculosis PncA, 1D NMR spectra were monitored at various molar ratios of PncA and PZA. The line-width of PZA was changed as PncA was added into PZA with different molar ratios. These results suggested that determination of PncA enzymatic activity could potentially serve as an indirect measure of PZA susceptibility.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.462-465
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• 2006

An important issue in the oxidation of pentachlorophenol (PCP) by the enzyme horseradish peroxidase (HRP) is enzyme inactivation during the reaction. This study was initiated to investigate the ability of two nonionic surfactants (Tween 20 and Tween 80) to mitigate HRP inactivation. The surfactants were tested at concentrations below and above their critical micelle concentrations (CMCs). Enhancement of PCP oxidation was observed at sub-CMCs, indicating effective protection of HRP by the two surfactants. Maximum levels of PCP removal were observed when the concentrations of Tween 20 and Tween 80 were 40 and 50% of the CMCs, respectively At supra-CMCs, both surfactants caused a noticeable reduction in the extent of PCP removal.

• BMB Reports
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• v.51 no.12
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• pp.609-610
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• 2018

Glycosylation is one form of protein modification and plays a key role in protein stability, function, signaling regulation and even cancer. NleB and SseK are bacterial effector proteins and possess glycosyltransferase activity, even though they have different substrate preferences. NleB/SseKs transfer the GlcNAc sugar to an arginine residue of host proteins, leading to reduced $NF-{\kappa}B-dependent$ responses. By combining X-ray crystallography, NMR, molecular dynamics, enzyme kinetic assays and in vivo experiments, we demonstrated that a conserved HEN (His-Glu-Asn) motif in the active site plays a key role in enzyme catalysis and virulence. The lid-domain regulates the opening and closing of the active site and the HLH domain determines the substrate specificity. Our findings provide evidence for the enzymatic mechanism by which arginine can be glycosylated by SseK/NleB enzymes.