Residue N84 of Yeast Cystathionine β-Synthase is a Determinant of Reaction Specificity

Cystathionine β-synthase (CBS) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue N84 of yeast CBS (yCBS), predicted to form a hydrogen bond with the hydroxyl moiety of the PLP cofactor, was mutated to alanine, aspartate and histidine. The truncated form of yCBS (ytCBS, residues 1-353) was employed in this study to eliminate any effects of the C-terminal, regulatory domain. The k_cat/K_m^l-Ser of the N84A, N84D and N84H mutants for the β-replacement reaction is reduced by a factor of 230, 11000 and 640, respectively. Fluorescence resonance energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and N84A mutant, is altered in N84H and absent in N84D. PLP saturation values of 73%, 30% and 67% were observed for the alanine, aspartate and histidine mutants, respectively, compared to 98% for the wild-type enzyme. A marginal β-elimination activity was detected for N84D (k_cat/K_m^l-Ser = 0.23 ± 0.02 M^-1 s^-1) and N84H (k_cat/K_m^l-Ser = 0.34 ± 0.06 M^-1 s^-1), in contrast with wild-type ytCBS and the N84A mutant, which do not catalyze this reaction. The ytCBS-N84D enzyme is also inactivated upon incubation with l-serine, via an aminoacrylate-mediated mechanism. These results demonstrate that residue N84 is essential in maintaining the orientation of the pyridine ring of the PLP cofactor and the equilibrium between the open and closed conformations of the active site.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k_cat′ for desulfination of l-cysteine sulfinate increased to 0.5 s^− 1 (from 0.05 s^− 1 in wild-type enzyme), whereas k_cat′ for transamination of the same substrate was reduced from 510 s^− 1 to 0.05 s^− 1. Similarly, k_cat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s^− 1 to 0.07 s^− 1, whereas k_cat′ for transamination was reduced from 530 s^− 1 to 0.13 s^− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.     

Investigation of the interaction of gold(III)-alkyldiamine complexes with l-histidine and imidazole ligands by H and C NMR, and UV spectrophotometry

Reactions of Au(III)-alkyldiamine complex with l-histidine and imidazole were carried out and monitored time-dependant by ¹H and ¹³C NMR. Kinetics for the [Au(en)Cl₂]⁺ reaction with l-histidine was determined by initial rate method at constant pH and 25 °C using UV-Vis absorption technique, and found to be first order with respect to each component, with a pseudo second order rate constant of 39 ± 3 M⁻¹ s⁻¹. Reaction rates of l-histidine and imidazole reactions with the [Au(en)Cl₂]⁺ complex was found to be strongly dependant on pD. The pD also has profound effect on the stability of the complex. It was observed that concurrent redox reactions also take place in solution in which Au(III) is reduced to metallic Au(0), while l-histidine and imidazole are oxidized to oxy and hydroxyl products. The optimization of the structure of [(His)Au(en)]³⁺ complex was carried out by gaussian03 at the RB3LYP level that showed a distorted square pyramid with the histidine carboxyl group at the pyramid top.     

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ± 4 μM, K_Mg^2 + = 294 ± 22 μM, K_L-gln = 11 ± 1 mM, and k_cat = 2.2 ± 0.2 s^− 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.     

Novel enzymatic activity derived from the Semliki Forest virus capsid protein

The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and k_cat/K_M values up to 5 × 10⁵ s⁻¹ M⁻¹. The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pK_a1 = 6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k₂ = 4.4±0.6 s⁻¹ and k₃ = 1.6±0.5 s⁻¹, respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities.     

Insight into the reaction mechanism of the Escherichia coli cyclopropane fatty acid synthase: Isotope exchange and kinetic isotope effects

Cyclopropanation of unsaturated lipids is an intriguing enzymatic reaction and a potential therapeutic target in Mycobacterium tuberculosis. Cyclopropane fatty acid synthase from Escherichia coli is the only in vitro model available to date for mechanistic and inhibition studies. While the overall reaction mechanism of this enzymatic process is now well accepted, some mechanistic issues are still debated. Using homogeneous E. coli enzyme we have shown that, contrary to previous report based on in vivo experiments, there is no exchange of the cylopropane methylene protons with the solvent during catalysis, as probed by ultra high resolution mass spectrometry. Using [methyl-¹⁴C]-labeled and [methyl-³H₃]-S-adenosyl-l-methionine we have measured a significant intermolecular primary tritium kinetic isotope effect (^TV/K_app = 1.8 ± 0.1) consistent with a partially rate determining deprotonation step. We conclude that both chemical steps of this enzymatic cyclopropanation, the methyl addition onto the double bond and the deprotonation step, are rate determining, a common situation in efficient enzymes.     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the k_cat/K_M value of CITase-598K was approximately two times higher (32.2 s^− 1 mM^− 1) than that of CITase-T3040 (17.8 s^− 1 mM^− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Identification, expression, and characterization of a novel bacterial RGI lyase enzyme for the production of bio-functional fibers

A gene encoding a putative rhamnogalacturonan I (RGI) Lyase (EC 4.2.2.-) from Bacillus licheniformis (DSM13) was selected after a homology search and phylogenetic analysis and optimized with respect to codon usage. The designed gene was transformed into Pichia pastoris and the enzyme was produced in the eukaryotic host with a high titer in a 5 l bioreactor. The RGI Lyase was purified by Cu²⁺ affinity chromatography and 1.1 g pure enzyme was achieved pr. L. When the denatured protein was deglycosylated with EndoH, the molecular weight of the protein decreased to 65 kDa, which correlated with the predicted molecular weight of the mature RGI Lyase of 596 amino acids. By use of a statistical design approach, with potato rhamnogalacturonan as the substrate, the optimal reaction conditions for the RGI Lyase were established to be: 61 °C, pH 8.1, and 2 mM of both Ca²⁺ and Mn²⁺ (specific activity 18.4 U/mg; K_M 1.2 mg/ml). The addition of both Ca²⁺ and Mn²⁺ was essential for enzyme activity. The enzyme retained its catalytic activity at higher temperatures and the enzyme has a half life at 61 °C of 15 min. The work thus demonstrated the workability of in silico based screening coupled with a synthetic biology approach for gene synthesis for identification and production of a thermostable enzyme.     

Cannibalism contributes significantly to the diet of cultured sand crabs, Portunus pelagicus (L.): A dual stable isotope study

The significance of cannibalism in the diet of juvenile pond-cultured blue swimmer crabs (Portunus pelagicus (L.)) was investigated using dual stable isotope analysis of carbon and nitrogen. In a laboratory feeding experiment, δ¹⁵N demonstrated a constant trophic shift (Δδ¹⁵N ≈+ 1.6‰), and therefore seemed to be a reliable indicator for assessing trophic position for P. pelagicus. This agrees with previously reported trends. Difference in growth rate did not seem to influence δ¹⁵N values. In contrast, δ¹³C did not display consistent shifts between trophic levels (range of Δδ¹³C: + 1 to + 1.7‰). The results from the pond experiment showed that larger individuals had a more enriched δ¹⁵N than smaller individuals, which, when compared to the results from the laboratory experiment, indicates that larger individuals were at a higher trophic level. This is most likely due to cannibalism prevailing in the pond rather than a direct result of faster growth rate. Cannibalistic behaviour might further increase growth, resulting in the observed positive correlation between size and δ¹⁵N.     

The nitrite-oxidizing community in activated sludge from a municipal wastewater treatment plant determined by fatty acid methyl ester-stable isotope probing

Metabolically-active autotrophic nitrite oxidizers from activated sludge were labeled with ¹³C-bicarbonate under exposure to different temperatures and nitrite concentrations. The labeled samples were characterized by FAME-SIP (fatty acid methyl ester-stable isotope probing). The compound cis-11-palmitoleic acid, which is the major lipid of the most abundant nitrite oxidizer in activated sludge, Candidatus Nitrospira defluvii, showed ¹³C-incorporation in all samples exposed to 3 mM nitrite. Subsequently, the lipid cis-7-palmitoleic acid was labeled, and it indicated the activity of a nitrite oxidizer that was different from the known Nitrospira taxa in activated sludge. The highest incorporation of cis-7-palmitoleic acid label was found after incubation with a nitrite concentration of 0.3 mM at 17 and 22 °C. While activity of Nitrobacter populations could not be detected by the FAME-SIP approach, an unknown nitrite oxidizer with the major lipid cis-9 isomer of palmitoleic acid exhibited ¹³C-incorporation at 28 °C with 30 mM nitrite. These results indicated flexibility of nitrite-oxidizing guilds in a complex community responding to different conditions. Labeled lipids so far not described for activated sludge-associated nitrifiers indicated the presence of unknown nitrite oxidizers in this habitat. The FAME-SIP-based information can be used to define appropriate conditions for the enrichment of nitrite-oxidizing guilds from complex samples.     

Conformational changes and reaction of clostridial glycosylating toxins

The crystal structures of the catalytic fragments of ‘lethal toxin’ from Clostridium sordellii and of ‘α-toxin’ from Clostridium novyi have been established. Almost half of the residues follow the chain fold of the glycosyl-transferase type A family of enzymes; the other half forms large α-helical protrusions that are likely to confer specificity for the respective targeted subgroup of Rho proteins in the cell. In the crystal, the active center of α-toxin contained no substrates and was disassembled, whereas that of lethal toxin, which was ligated with the donor substrate UDP-glucose and cofactor Mn^2 +, was catalytically competent. Surprisingly, the structure of lethal toxin with Ca^2 + (instead of Mn^2 +) at the cofactor position showed a bound donor substrate with a disassembled active center, indicating that the strictly octahedral coordination sphere of Mn^2 + is indispensable to the integrity of the enzyme. The homologous structures of α-toxin without substrate, distorted lethal toxin with Ca^2 + plus donor, active lethal toxin with Mn^2 + plus donor and the homologous Clostridium difficile toxin B with a hydrolyzed donor have been lined up to show the geometry of several reaction steps. Interestingly, the structural refinement of one of the three crystallographically independent molecules of Ca^2 +-ligated lethal toxin resulted in the glucosyl half-chair conformation expected for glycosyl-transferases that retain the anomeric configuration at the C1″ atom. A superposition of six acceptor substrates bound to homologous enzymes yielded the position of the nucleophilic acceptor atom with a deviation of < 1 Å. The resulting donor-acceptor geometry suggests that the reaction runs as a circular electron transfer in a six-membered ring, which involves the deprotonation of the nucleophile by the β-phosphoryl group of the donor substrate UDP-glucose.     

New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate

N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-d-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-d-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117 ± 2 U mg⁻¹ at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the K_m for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.     

SEA domain autoproteolysis accelerated by conformational strain: mechanistic aspects

A subclass of SEA (sea urchin sperm protein, enterokinase, and agrin) domain proteins undergoes autoproteolysis between glycine and serine in a conserved G^− 1S^+ 1VVV motif to generate stable heterodimers. Autoproteolysis has been suggested to involve only the intramolecular catalytic action of the conserved serine hydroxyl in combination with conformational strain of the glycine-serine peptide bond. We conducted a number of experiments and simulations on the SEA domain from the MUC1 mucin to test this mechanism. Alanine-scanning mutagenesis of polar residues in the vicinity of the cleavage site demonstrates that only the nucleophile at position + 1 is required for efficient proteolysis. Molecular modeling shows that an uncleaved trans peptide is incompatible with the native heterodimeric structure, resulting in disruption of secondary structure elements and distortion of the scissile peptide bond. Insertion of glycine residues (to obtain G_nG^− 1S^+ 1VVV motifs) appears to relieve strain, and autoproteolysis is 100 times slower in a 1G (n = 1) mutant and not measurable in 2G and 4G mutants. Removal of the catalytic serine hydroxyl hampers cleavage considerably, but measurable autoproteolysis of this S1098A mutant still proceeds in the presence of strain alone. The uncleaved SEA precursor populates interconverting partially folded conformations, and autoproteolysis coincides with adoption of proper β-sheet secondary structure and completed folding. Molecular dynamics simulations of the precursor show that the serine hydroxyl and the preceding glycine carbonyl carbon can be in van der Waals contact at the same time as the scissile peptide bond becomes strained. These observations are all consistent with autoproteolysis accelerated by N → O acyl shift and conformational strain imposed upon protein folding in a reaction for which the free-energy barrier is decreased by substrate destabilization rather than by transition-state stabilization. The energetics of this coupled folding and autoproteolysis mechanism is accounted for in an accompanying article.     

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: High-affinity Zn-binding site at the P-side of the membrane

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn²⁺. Inhibition of liposome-bound bovine cytochrome oxidase by external Zn²⁺ titrates with a K_i of 1 ± 0.3 μM. Presumably, the Zn²⁺-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu_B is tentatively proposed to be responsible for the effect.     

A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4–1.2 pH units, likely due to repositioning of the cofactor and modification of the pK_a of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3–3, 26–58 and 124–568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The K_m^l-Cth of E333 substitution variants is increased ~ 17-fold, while K_m^l-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (k_cat/K_m^l-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (k_cat/K_m^l-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (k_cat/K_m^l-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca²⁺, Ba²⁺, and Zn²⁺ in a concentration-dependent manner, while it is dramatically inhibited by Hg²⁺. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.