Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

This study investigated the functional roles of the N-terminal Ca²⁺ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca²⁺-binding site of PAD4 were mutated to disrupt the binding of Ca²⁺ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k _cat/K _m,BAEE values were 0.02, 0.63 and 0.01 s⁻¹mM⁻¹ (20.8 s⁻¹mM⁻¹ for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k _cat value of 0.3 s⁻¹ (13.3 s⁻¹ for wild-type), whereas D176A retained some catalytic power with a k _cat of 9.7 s⁻¹. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k _cat/K _m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca²⁺ indicated that the conformational stability of the enzyme is highly dependent on Ca²⁺ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca²⁺ ions in the N-terminal Ca²⁺-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca²⁺ ions play critical roles in the full activation of the PAD4 enzyme.     

Cloning, Expression, and Enzymatic Characterization of Isocitrate Dehydrogenase from Helicobacter pylori

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k _cat of 87 s^?1, K _m of 124 μM and k _cat/K _m of 7 × 10⁵ M^?1s^?1 toward isocitrate, k _cat of 80 s^?1, K _m of 176 μM and k _cat/K _m of 4.5 × 10⁵ M^?1s^?1 toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 °C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection.     

Functional role of dimerization of human peptidylarginine deiminase 4 (PAD4)

Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²⁺-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K _d) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K _d values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k _cat values than that of WT (13.4 s⁻¹). The K _d values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k _cat values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k _cat values of these interface mutants decreased as the K _d values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.     

Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemi l-asparaginase

During the manufacture of the chemotherapeutic enzyme Erwinia chrysanthemi l-asparaginase, a small proportion (approximately 5–15%) of acidic variants, including deamidated species, are observed. Although the deamidated forms appear to have similar specific activity and quaternary structure to the unmodified enzyme, monitoring and control of these forms is important from a regulatory perspective. The extent of Asn to Asp deamidation directly correlates with the time taken to thaw the Erwinia cells. Erwinia l-asparaginase is a tetrameric enzyme containing one site, Asn₂₈₁, theoretically very labile to deamidation due to the sequence Asn-Gly. Structurally, this part of the protein sequence is completely buried inside the tetramer, but solvent-exposed upon tetramer dissociation. During the cell thawing and alkaline lysis sequence of the process, lengthening the cell thaw times by up to 24 h allowed tetramer to reassociate, protected Asn₂₈₁ from deamidation and reduced the acidic species content of the l-asparaginase from approximately 17% to 9% as measured by weak cation-exchange (WCX) HPLC. The correlation of cell thaw time with acidic species content was also confirmed using capillary zone electrophoresis (CZE) and peptide mapping. These studies demonstrate that cell thaw time is an important, if unexpected, control variable for l-asparaginase deamidation.     

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis,spectroscopic, and computational methods

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV–vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k_cat = 3.9(4) s^? 1) lower than that of wt SmNir (k_cat = 240(50) s^? 1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N^δ1H…OC hydrogen bond that partially shortcuts the T1–T2 bridging Cys-His covalent pathway. The role of the N^δ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH 6 and 10 by UV–vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1–T2 bridging chemical pathway.     

GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509

A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K _m and k _cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s^?1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K _m and k _cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s^?1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K _m and k _cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s^?1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.     

Methanothermobacter thermautotrophicus tRNA Gln confines the amidotransferase GatCAB to asparaginyl-tRNA Asn formation

Many prokaryotes form the amide aminoacyl-tRNAs glutaminyl-tRNA and asparaginyl-tRNA by tRNA-dependent amidation of the mischarged tRNA species, glutamyl-tRNA^Gln or aspartyl-tRNA^Asn. Archaea employ two such amidotransferases, GatCAB and GatDE, while bacteria possess only one, GatCAB. The Methanothermobacter thermautotrophicus GatDE is slightly more efficient using Asn as an amide donor than Gln (k_cat/K_M of 5.4 s⁻¹/mM and 1.2 s⁻¹/mM, respectively). Unlike the bacterial GatCAB enzymes studied to date, the M. thermautotrophicus GatCAB uses Asn almost as well as Gln as an amide donor (k_cat/K_M of 5.7 s⁻¹/mM and 16.7 s⁻¹/mM, respectively). In contrast to the initial characterization of the M. thermautotrophicus GatCAB as being able to form Asn-tRNA^Asn and Gln-tRNA^Gln, our data demonstrate that while the enzyme is able to transamidate Asp-tRNA^Asn (k_cat/K_M of 125 s⁻¹/mM) it is unable to transamidate M. thermautotrophicus Glu-tRNA^Gln. However, M. thermautotrophicus GatCAB is capable of transamidating Glu-tRNA^Gln from H. pylori or B. subtilis, and M. thermautotrophicus Glu-tRNA^Asn. Thus, M. thermautotrophicus encodes two amidotransferases, each with its own activity, GatDE for Gln-tRNA and GatCAB for Asn-tRNA synthesis.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

Structural and Functional Roles of Glycosylation in Fungal Laccase from Lentinus sp.

Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O₂ to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn⁷⁵, Asn²³⁸, and Asn⁴⁵⁸ were elucidated. Initial rate kinetic analysis revealed that the k _cat, K _m, and k _cat/K _m of nLcc4 with substrate ABTS were 3,382 s ^-1, 65.0 ± 6.5 μM, and 52 s ^-1μM^-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s ^-1, 56.7 ± 3.2 μM, and 0.004 s ^-1μM^-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids

Leukotriene A₄ hydrolase (LTA4H––EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA₄ through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k _cat/K _m values). Among them, the benzyl ester of aspartic acid exhibited a k _cat/K _m value that was more than two orders of magnitude higher (1.75 × 10⁵ M^?1 s^?1) as compared to l-Arg (1.5 × 10³ M^?1 s^?1). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H.     

Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k _cat and k _cat/K _m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K _i values for d-glucose (83.3–357 mM) and d-xylose (15.6–70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K _t ^0.5?～?45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k _cat and k _cat/K _m on natural substrates xylobiose (407 s^?1, 138 s^?1?mM^?1), xylotriose (235 s^?1, 80.8 s^?1?mM^?1), and xylotetraose (146 s^?1, 32.6 s^?1?mM^?1).     

Purification and Characterization of Exo-Inulinase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. d9 Strain

This study intended to purify and characterise exo-inulinase of diesel-degrading Paenibacillus sp. D9. The whole genome sequencing of Paenibacillus sp. D9 revealed to possess the sacC gene that is encoded as exo-inulinase/levanase. This isolate was capable of producing a maximum of 50.9 IU/mL of exo-inulinase activity within 3 days at 30?°C, 200 rpm and pH of 7.0 on minimal salt medium agar supplemented with 1% (w/v) inulin. An exo-inulinase of 58.5 kDa was purified using ammonium sulphate precipitation, HiTrap QFF column and MMC column chromatographies with a specific activity of 4333 IU/mg, 7.1% recovery and a 4.3-fold increase in purity. The purified D9 exo-inulinase had temperature and pH optimum at 40?°C and pH 4.0, respectively, with the Michaelis constant of 5.5 mM and a maximal velocity of 476.2 IU/mg, respectively. Catalytic constant, k _cat was calculated to be 42.6 s^?1 with a catalytic efficiency (k _cat /K _m ) of 7.6 s^?1 mM^?1. The presence of Ca²⁺ enhanced the activity of D9 exo-inulinase while Hg²⁺ completely inhibited the activity, other compounds such as Fe³⁺ and Cu²⁺ had an inhibitory effect. The results of amino acid alignment and the complete degradation of inulin into fructose by the purified enzyme confirmed that inulinase from Paenibacillus sp. D9 is an exo-form. The phylogenetic tree based on the protein sequences indicates that bacterial exo-inulinases possess a common ancestry.     

Cloning of Intron-Removed Enolase Gene and Expression,Purification, Kinetic Characterization of the Enzyme from Theileria annulata

Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni–NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K _m: 106 μM, k_cat: 37 s^?1, and k _cat/K _m: 3.5 × 10⁵ M^?1 s^?1. These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.     

Cloning,functional expression,biochemical characterization,and structural analysis of a haloalkane dehalogenase from <Emphasis Type="Italic">Plesiocystis pacifica</Emphasis> SIR-1

A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 Å resolution to an R-factor of 21.93%. The enzyme is composed of an α/β-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37°C with a pH optimum between pH 8 and 9. The K_m and k_cat values for 1-bromobutane were 24.0 mM and 8.08 s^?1. Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

Long-range interaction between the enzyme active site and a distant allosteric site in the human mitochondrial NAD(P)-dependent malic enzyme

Our previous study has suggested that mutation of the amino acid residue Asp102 has a significant effect on the fumarate-mediated activation of human mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD(P)-ME). In this paper, we examine the cationic amino acid residue Arg98, which is adjacent to Asp102 and is highly conserved in most m-NAD(P)-MEs. A series of R98/D102 mutants were created to examine the possible interactions between Arg98 and Asp102 using the double-mutant cycle analysis. Kinetic analysis revealed that the catalytic efficiency of the enzyme was severely affected by mutating both Arg98 and Asp102 residues. However, the binding energy of these mutant enzymes to fumarate as determined by analysis of the K_A,Fum values, show insignificant differences, indicating that the mutation of Arg98 and Asp102 did not cause a significant decrease in the binding affinity of fumarate. The overall coupling energies for R98K/D102N as determined by analysis of the k_cat/K_m and K_A,Fum values were −2.95 and −0.32 kcal/mol, respectively. According to these results, we conclude that substitution of both Arg98 and Asp102 residues has a synergistic effect on the catalytic ability of the enzyme.     

Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex

The latex from Vasconcellea quercifolia (“oak leaved papaya”), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K _m = 0.454 ± 0.046 mM, k _cat = 1.57 ± 0.07 s^?1, k _cat/K _m = 3.46 × 10³ ± 14 s^?1 M^?1). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the “papain-like” family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.