Structural basis for catalysis and substrate specificity of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase

N-Carbamoyl-d-amino acid amidohydrolase is an industrial biocatalyst to hydrolyze N-carbamoyl-d-amino acids for producing valuable d-amino acids. The crystal structure of N-carbamoyl-d-amino acid amidohydrolase in the unliganded form exhibits a alpha-beta-beta-alpha fold. To investigate the roles of Cys172, Asn173, Arg175, and Arg176 in catalysis, C172A, C172S, N173A, R175A, R176A, R175K, and R176K mutants were constructed and expressed, respectively. All mutants showed similar CD spectra and had hardly any detectable activity except for R173A that retained 5% of relative activity. N173A had a decreased value in kcat or Km, whereas R175K or R176K showed high Km and very low kcat values. Crystal structures of C172A and C172S in its free form and in complex form with a substrate, along with N173A and R175A, have been determined. Analysis of these structures shows that the overall structure maintains its four-layer architecture and that there is limited conformational change within the binding pocket except for R175A. In the substrate-bound structure, side chains of Glu47, Lys127, and C172S cluster together toward the carbamoyl moiety of the substrate, and those of Asn173, Arg175, and Arg176 interact with the carboxyl group. These results collectively suggest that a Cys172-Glu47-Lys127 catalytic triad is involved in the hydrolysis of the carbamoyl moiety and that Arg175 and Arg176 are crucial in binding to the carboxyl moiety, hence demonstrating substrate specificity. The common (Glu/Asp)-Lys-Cys triad observed among N-carbamoyl-d-amino acid amidohydrolase, NitFhit, and another carbamoylase suggests a conserved and robust platform during evolution, enabling it to catalyze the reactions toward a specific nitrile or amide efficiently.     

Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement

A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k _cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K _i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

     

Kinetic and structural characterization of Slr0077/SufS, the essential cysteine desulfurase from Synechocystis sp. PCC 6803

Cysteine desulfurases, designated NifS, IscS, and SufS, cleave L-cysteine to form alanine and an enzyme cysteinyl persulfide intermediate. Genetic studies on the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 have shown that of the three Nif/Isc/SufS-like proteins encoded in its genome only the sequence group II protein, Slr0077/SufS, is essential. This protein has been overexpressed in Escherichia coli, purified to homogeneity, shown to bind pyridoxal-5'-phosphate (PLP) and to catalyze cysteine desulfuration, and characterized in terms of its structure and kinetics. The results suggest that catalysis in the absence of accessory factors has two constituent pathways, one involving nucleophilic attack by C372 to form the Slr0077/SufS-bound cysteinyl persulfide intermediate and the second involving intermolecular attack by the sulfur of a second molecule of the substrate on the initial l-cysteine-PLP complex to form free l-cysteine persulfide. The second pathway is operant in the C372A variant protein, explaining why it retains significant activity, which is proportional to the concentration of l-cysteine (i.e., does not saturate). C-S bond cleavage by the first (normal) pathway is considerably less efficient than the equivalent step in a group I desulfurase (Slr0387) from the same organism (characterized in the accompanying paper). The 1.8 A crystal structure of the protein, which is very similar to that previously reported for E. coli SufS, shows that the loop on which C372 resides is well-ordered and shorter by 11 residues than the corresponding disordered loop of the group I NifS-like protein from Thermotoga maritima. Sequence comparisons establish that the T. maritima and Slr0387 proteins have loops of similar length. The combined structural and kinetic data imply that the modest activity of Slr0077/SufS and other SufS proteins in comparison to their sequence group I (NifS/IscS-like) paralogues results from inefficiency in the nucleophilic attack step associated with differences in the structure or dynamics of this loop. The recent reports that SufS proteins can be activated manyfold by binding to SufE thus implies that the accessory protein either accelerates nucleophilic attack by the conserved cysteine residue of SufS by a conformational mechanism or itself contributes a nucleophilic cysteine for more efficient intermolecular attack.     

Pre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease

Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5′ side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3′-5′-exonuclease, 3′-phosphodiesterase, and 3′-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre–steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding–site residue, Arg181, was analyzed too; it changed its conformation when enzyme–substrate and enzyme–product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction.     

Maize phosphoenolpyruvate carboxylase. Mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation

Phosphoenolpyruvate carboxylases (PEPC, EC 4.1.1.31) from higher plants are regulated by both allosteric effects and reversible phosphorylation. Previous x-ray crystallographic analysis of Zea mays PEPC has revealed a binding site for sulfate ion, speculated to be the site for an allosteric activator, glucose 6-phosphate (Glc-6-P) (Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K., and Kai, Y. (2002) Structure (Lond.) 10, 1721-1730). Because kinetic experiments have also supported this notion, each of the four basic residues (Arg-183, -184, -231, and -372' on the adjacent subunit) located at or near the binding site was replaced by Gln, and the kinetic properties of recombinant mutant enzymes were investigated. Complete desensitization to Glc-6-P was observed for R183Q, R184Q, R183Q/R184Q (double mutant), and R372Q, as was a marked decrease in the sensitivity for R231Q. The heterotropic effect of Glc-6-P on an allosteric inhibitor, l-malate, was also abolished, but sensitivity to Gly, another allosteric activator of monocot PEPC, was essentially not affected, suggesting the distinctness of their binding sites. Considering the kinetic and structural data, Arg-183 and Arg-231 were suggested to be involved directly in the binding with phosphate group of Glc-6-P, and the residues Arg-184 and Arg-372 were thought to be involved in making up the site for Glc-6-P and/or in the transmission of an allosteric regulatory signal. Most unexpectedly, the mutant enzymes had almost lost responsiveness to regulatory phosphorylation at Ser-15. An apparent lack of kinetic competition between the phosphate groups of Glc-6-P and of phospho-Ser at 15 suggested the distinctness of their binding sites. The possible roles of these Arg residues are discussed.     

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding constant decreases, leading to an increase in K(M)m. The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V(M)max. The small difference between the absolute values of V(M)max suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.     

Subunit interface residues of glutathione S-transferase A1-1 that are important in the monomer-dimer equilibrium

Alpha class glutathione S-transferase, isozyme A1-1, is a dimer (51 kDa) of identical subunits. Using the crystal structure, two main areas of subunit interaction were chosen for study: (1) the hydrophobic ball and socket comprised of Phe52 from one subunit fitting into a socket formed on the other subunit by Met94, Phe136, and Val139 and (2) the Arg/Glu region consisting of Arg69 and Glu97 from both subunits. We introduced substitutions of these residues, by site-directed mutagenesis, to evaluate the importance of each at the subunit interface and to determine if monomeric enzymes could be generated using single mutations. Mutating each residue of the socket region to alanine results in little change in the kinetic parameters, and all are dimeric enzymes. In contrast, when Phe52, the ball residue, is replaced with alanine, the enzyme has very low activity and a weight average molecular mass of 31.9 kDa, as determined by sedimentation equilibrium experiments. Substitutions for Glu97 which eliminate the charge cause no appreciable changes in the kinetic parameters or molecular mass. Eliminating the charge on Arg69 (as in R69Q) results in a dimeric enzyme; however, when the charge is reversed (as in R69E), the weight average molecular mass is greatly shifted toward that of the monomer (33 kDa) and the changes in kinetic parameters are reasonably small. We determined the molecular masses in the presence of glutathione for F52A and R69E to ascertain whether the monomeric species retains activity. For R69E, it appears that the monomer is active, albeit less so than the dimer, while for F52A, the monomer and dimer both appear to exhibit very low activity. The dimeric species is needed to obtain high specific activity. We conclude that, of the residues that were studied, Phe52 and Arg69 are the major determinants of dimer formation and a single mutation at either position substantially hinders dimerization. The use of a mutant glutathione S-transferase which retains activity yet has a greatly weakened tendency to dimerize (such as R69E) may be advantageous for certain applications of GST fusion proteins.     

A new inhibitor design strategy for carboxypeptidase A as exemplified by N-(2-chloroethyl)-N-methylphenylalanine

N-(2-Chloroethyl)-N-methylphenylalanine was designed and synthesized in an optically active form as a novel class of mechanism-based inactivator for carboxypeptidase A (CPA). It was anticipated that the chloroethylamino moiety of the CPA bound inhibitor undergoes an intramolecular SN2 reaction to generate a chemically reactive species (an aziridinium ion) which is expectedly subjected to a nucleophilic attack by the carboxylate of Glu-270, leading to covalent modification of the carboxylate. The irreversible nature of the inhibition of CPA by the inhibitor was supported by the kinetic data: the enzyme lost its enzymic activity in a time-dependent manner in the presence of the inhibitor and the inactivated CPA failed to regain the activity upon dialysis. Interestingly, the (R)-isomer that belongs to the D-series was more potent than its enantiomer.     

Tyr212: a key residue involved in strand discrimination by the DNA mismatch repair endonuclease MutH

The molecular mechanism of how the dam-methylation status of the DNA is recognized during DNA mismatch repair by the strand discrimination endonuclease MutH is not known. A comparison of the crystal structure of MutH with those of co-crystal structures of several restriction endonucleases, together with a multiple sequence alignment of MutH and related proteins suggested that Phe94, Arg184 and Tyr212 could be involved in discrimination between a methylated or unmethylated adenine in the d(GATC) sequence. A mutational analysis revealed that the variants R184A and Y212S, but not F94A, were substantially reduced in their ability to complement a mismatch repair deficiency in a mutH(-) Escherichia coli strain. In vitro, R184A displayed a strongly reduced endonuclease activity, whereas the Y212S variant has almost completely lost its preference for cleaving the unmethylated strand at hemimethylated d(GATC) sites. Furthermore, the Y212 variant can cleave fully methlyated d(GATC) sites at a comparable rate to unmethylated d(GATC) sites. This demonstrates that Tyr212 is an important, if not the only amino acid residue in MutH for sensing the methylation status of the DNA.     

Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants.

Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady-state and transient-state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt-bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second-order rate constants for compound I reduction (k2app), and decreased first-order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron-transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.     

Understanding how the distal environment directs reactivity in chlorite dismutase: spectroscopy and reactivity of Arg183 mutants

The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O(2) and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O-O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV-visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pK(a) values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pK(a) of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid-base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.     

Arginine residues in the active site of human phenol sulfotransferase (SULT1A1)

Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg78, Arg130, and Arg257 may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg78 is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg78 is in close proximity to the site of sulfuryl group transfer. Arg257 is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Site-directed mutagenesis demonstrated that Arg257 is critical for SULT1A1: both R257A and R257E are inactive. Although Arg130 is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg130 is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg257, and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.     

Replacement of thrombin residue G184 with Lys or Arg fails to mimic Na+ binding

Na+ binding to thrombin enhances the catalytic activity toward numerous synthetic and natural substrates. The bound Na+ is located in a solvent channel 16 A away from the catalytic triad, and connects with D189 in the S1 site through an intervening water molecule. Molecular modeling indicates that the G184K substitution in thrombin positions the protonated epsilon-amino group of the Lys side-chain to replace the bound Na+. Likewise, the G184R substitution positions the guanidinium group of the longer Arg side-chain to replace both the bound Na+ and the connecting water molecule to D189. We explored whether the G184K or G184R substitution would replace the bound Na+ and yield a thrombin derivative stabilized in the highly active fast form. Both the G184K and G184R mutants lost sensitivity to monovalent cations, as expected, but their activity toward a chromogenic substrate was compromised up to 200-fold as a result of impaired diffusion into the S1 site and decreased deacylation rate. Interestingly, both G184K and G184R substitutions compromised cleavage of procoagulant substrates fibrinogen and PAR1 more than that of the anticoagulant substrate protein C. These findings demonstrate that Na+ binding to thrombin is difficult to mimic functionally with residue side-chains, in analogy with results from other systems.     

Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance

The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K(2)TeO(3) at 128 microg/ml, compared to the MIC of 2 microg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a approximately 20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.     

Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase

There is now overwhelming evidence supporting a common mechanism for fumarate reduction in the respiratory fumarate reductases. The X-ray structures of substrate-bound forms of these enzymes indicate that the substrate is well positioned to accept a hydride from FAD and a proton from an arginine side chain. Recent work on the enzyme from Shewanella frigidimarina [Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2000) Biochemistry 39, 10695-10701] has strengthened the assignment of an arginine (Arg402) as the proton donor in fumarate reduction. Here we describe the crystallographic and kinetic analyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme. The crystal structure of the R402A mutant (2.0 A resolution) shows it to be virtually identical to the wild-type enzyme, apart from the fact that a water molecule occupies the position previously taken by part of the guanidine group of R402. Although structurally similar to the wild-type enzyme, the R402A mutant is inactive under all the conditions that were studied. This implies that a water molecule, in this position in the active site, cannot function as the proton donor for fumarate reduction. In contrast to the R402A mutation, both the R402K and R402Y mutant enzymes are active. Although this activity was at a very low level (at pH 7.2 some 10(4)-fold lower than that for the wild type), it does imply that both lysine and tyrosine can fulfill the role of an active site proton donor, albeit very poorly. The crystal structures of the R402K and R402Y mutant enzymes (2.0 A resolution) show that distances from the lysine and tyrosine side chains to the nearest carbon atom of fumarate are approximately 3.5 A, clearly permitting proton transfer. The combined results from mutagenesis, crystallographic, and kinetic studies provide formidable evidence that R402 acts as both a Lewis acid (stabilizing the build-up of negative charge upon hydride transfer from FAD to fumarate) and a Br?nsted acid (donating the proton to the substrate to complete the formation of succinate).     

Functional role of a conserved arginine residue located on a mobile loop of alkanesulfonate monooxygenase

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower k(cat)/K(m) value (0.04 ± 0.01 μM(-1) min(-1)) relative to wild-type (1.17 ± 0.22 μM(-1) min(-1)), there was no activity observed with the R297A SsuD variant. Each of the arginine variants had similar K(d) values for flavin binding as wild-type SsuD (0.32 ± 0.15 μM), but there was no measurable binding of octanesulfonate. The low levels of activity for the R297A and R297K SsuD variants correlated with the absence of any detectable C4a-(peroxy)flavin formation in stopped-flow kinetic studies. Single-turnover experiments were performed in the presence of SsuE to evaluate both the reductive and oxidative half-reaction. With wild-type SsuD a lag phase is observed following the reductive half-reaction by SsuE that represents flavin transfer or conformational changes associated with the binding of substrates. Evaluation of the Arg297 SsuD variants in the presence of SsuE showed no lag phase following reduction by SsuE, and the flavin was oxidized immediately following the reductive half-reaction. These results corresponded with a lack of detectable changes in the proteolytic susceptibility of R297A and R297K SsuD in the presence of reduced flavin and/or octanesulfonate, signifying the absence of a conformational change in these variants with the substitution of Arg297.     

Insights into the catalytic mechanism of human sEH phosphatase by site-directed mutagenesis and LC-MS/MS analysis

We have recently reported that human soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a novel phosphatase enzymatic activity. Based on a structural relationship with other members of the haloacid dehalogenase superfamily, the sEH N-terminal phosphatase domain revealed four conserved sequence motifs, including the proposed catalytic nucleophile D9, and several other residues potentially implicated in substrate turnover and/or Mg²⁺ binding. To enlighten the catalytic mechanism of dephosphorylation, we constructed sEH phosphatase active-site mutants by site-directed mutagenesis. A total of 18 mutants were constructed and recombinantly expressed in Escherichia coli as soluble proteins, purified to homogeneity and subsequently analysed for their kinetic parameters. A replacement of residues D9, K160, D184 or N189 resulted in a complete loss of phosphatase activity, consistent with an essential function for catalysis. In contrast, a substitution of D11, T123, N124 and D185 leads to sEH mutant proteins with altered kinetic properties. We further provide evidence of the formation of an acylphosphate intermediate on D9 by liquid chromatography-tandem mass spectrometry based on the detection of homoserine after NaBH₄ reduction of the phosphorylated enzyme, which identifies D9 as the catalytic nucleophile. Surprisingly, we could only show such homoserine formation using the D11N mutant, which strongly suggests D11 to be involved in the acylphosphate hydrolysis. In the D11 mutant, the second catalytic step becomes rate limiting, which then allows trapping of the labile intermediate. Substrate turnover in the presence of ¹⁸H₂O revealed that the nucleophilic attack during the second reaction step occurs at the acylphosphate phosphorous. Based on these findings, we propose a two-step catalytic mechanism of dephosphorylation that involves the phosphate substrate hydrolysis by nucleophilic attack by the catalytic nucleophile D9 followed by hydrolysis of the acylphosphate enzyme intermediate supported by D11.     

The enhanced affinity for thiolate anion and activation of enzyme-bound glutathione is governed by an arginine residue of human Mu class glutathione S-transferases

A series of chimeric human Mu class glutathione S-transferases were designed to determine mechanisms by which they activate enzyme-bound glutathione (GSH) for reaction with electrophilic substrates. In view of evidence that the His(107) residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y. V., Patskovska, L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-1202), the cognate Arg(107) residue of the hGSTM2 subunit was replaced (R107N or R107H) and arginine residues were also incorporated into position 107 of hGSTM1 (H107R) and hGSTM4 (S107R) subunits. The major distinguishing kinetic properties invariably associated with enzymes containing an Arg(107) residue include an inverse dependence of k(cat) on viscosity and lower K(m(GSH values relative to enzymes with other residues at that position. Moreover, affinities for GSH thiolate anion binding are greater for enzymes containing Arg(107))), with K(d) values of 20-50 microM that are consistent with the K(m(GSH values (10-25 microM) obtained by steady-state kinetic analyses. Both thermodynamic and kinetic and data indicate that the Arg(107))) residue is specifically involved in enhancing the binding affinity of GSH thiolate anion relative to that of the protonated form. These enzymes therefore, can be more effective at lower GSH concentrations. Combined mutations indicate that both Arg(107) and Tyr(6) residues are required for thiolate anion formation and stabilization. The three-dimensional structure of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that Arg(107) maintains an electrostatic interaction with the Asp(161) side chain (3 A apart), but is distant from the GSH-binding site. However, an alternative energetically favorable model places the guanidino group 4 A from the sulfur atom of bound GSH. It is suggested therefore, that in solution, motion of the positively charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the observed greater affinity of enzymes containing Arg(107) for binding of thiolate anion.     

The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities

Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.