Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.     

The molecular basis for inhibition of BphD, a C-C bond hydrolase involved in polychlorinated biphenyls degradation: large 3-substituents prevent tautomerization

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H>F>Cl>Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A.3-Cl HOPDA and S112A.3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A.HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A.3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.     

The tautomeric half-reaction of BphD, a C-C bond hydrolase. Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (lambda(max) is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (lambda(max) is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (lambda(max) is 506 nm) with a similar rate, 1/tau approximately 500 s(-1). The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 A away.     

Rhodococcus sp. R04 BphD催化C-C断裂的动力学分析

2-羟基-6-氧-6-苯基己-2,4-二烯酸水解酶（BphD）是一种多氯联苯微生物降解途径中的关键酶. 本文通过紫外-可见光光谱分别对突变酶S110A和H265A催化过程中酶-底物复合物进行检测,同时利用停流光谱技术对BphD及其突变酶（S110A、H265A和W266A）催化底物2-羟基-6-氧-6-苯基己-2,4-二烯酸（HOPDA）前稳态动力学进行了研究.结果表明,在BphD催化C-C断裂过程中,产物2-羟基戊-2,4-二烯酸（HPD）迅速生成,其速率常数为22 S-1. 底物的消耗（速率常数,22022 S-1和803 S-1）及酶-底物复合物的变化（速率常数,55556 S-1和664 S-1）表明该酶催化过程包括2个动力学阶段：快速底物酮基化作用和C-C键断裂过程.紫外-可见光光谱扫描结果显示,在突变酶S110A的催化过程中,酶-底物复合物在492 nm及510 nm处有最大光吸收,而在突变酶H265A催化中,却没有相似的光吸收,只是在480 nm产生1个新肩峰. BphD及其突变酶S110A、H265A和W266A动力学分析表明,Ser-110主要负责底物C-C键断裂;His-265负责底物由烯醇式向酮式转变,并且与Ser-110和Trp-266共同参与了随后的C-C键断裂过程. 结果揭示,除了传统的催化三联体（Ser-110,Asp-237,His-265）外,Trp-266在该水解酶催化反应中也发挥非常重要的作用,这一发现丰富了C-C水解酶的反应动力学机制.     

Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme

2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the alpha/beta hydrolase family. The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (Henderson, I. M. J., and Bugg, T. D. H. (1997) Biochemistry 36, 12252-12258), followed by stereospecific fragmentation. Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a (14)C-labeled substrate yielded a stoichiometry of <1% covalent intermediate, which could be accounted for by nonenzymatic processes. In contrast, incorporation of 5-6% of two atoms of (18)O from H(2)(18)O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate. Furthermore, time-dependent incorporation of (18)O from H(2)(18)O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl. These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine. The implication of this work is that the putative active site serine in this enzyme may have an alternative function, for example, as a base.     

Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants

C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110.     

The catalytic serine of meta-cleavage product hydrolases is activated differently for C-O bond cleavage than for C-C bond cleavage

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k(cat) value (6.3 ± 0.5 s(-1)) similar to that of HOPDA (6.5 ± 0.5 s(-1)). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k(cat)/K(m) values were independent of methanol concentration, while both k(cat) and K(m) values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k(obs) values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.     

Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase from the biphenyl degradation pathway

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.     

色氨酸残基在碳-碳水解酶催化中的关键作用

碳-碳水解酶（C-C水解酶）作为α/β水解酶超家族中的一员,负责催化环裂产物C-C键的断裂,该反应是细菌降解芳香族化合物途径中的关键步骤. 为了解水解酶的催化特性,本文对该酶部分氨基酸进行了定点突变,并对突变体的动力学参数,化学修饰剂对突变体活性的影响以及突变体的二级结构进行了测定.各突变体的动力学参数特征为：突变体S110A,H265A和D237A的催化效率为野生型的1/104~1/103;突变体W85A和W219A催化效率分别为野生型的5/18和1/3,而同为色氨酸的突变体,W266A的催化效率只有野生型的1/104. 化学修饰剂对突变体S110A,H265A,D237A和W266A的酶活性几乎没有影响;而对突变体W85A和W219A却有较大的影响,修饰后,其相对活性仅为对照的10%~30%. 突变体的圆二色谱（CD谱）分析表明,与野生型相比,突变体的二级结构没有发生改变. 证明了Ser110,Asp237,His265是2-羟基-6-氧-6-苯基己-2,4-二烯酸水解酶（HOPDA hydrolase, HOPDA水解酶）催化反应所必需的氨基酸,并提出了Trp266在催化反应中也同样起到了非常关键的作用.     

A new chemical mechanism catalyzed by a mutated aldehyde dehydrogenase.

NAD(P) aldehyde dehydrogenases (EC 1.2.1.3) are a family of enzymes that oxidize a wide variety of aldehydes into acid or activated acid compounds. Using site-directed mutagenesis, the essential nucleophilic Cys 149 in the NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli has been replaced by alanine. Not unexpectedly, the resulting mutant no longer shows any oxidoreduction phosphorylating activity. The same mutation, however, endows the enzyme with a novel oxidoreduction nonphosphorylating activity, converting glyceraldehyde 3-phosphate into 3-phosphoglycerate. Our study further provides evidence for an alternative mechanism in which the true substrate is the gem-diol entity instead of the aldehyde form. This implies that no acylenzyme intermediate is formed during the catalytic event. Therefore, the mutant C149A is a new enzyme which catalyzes a distinct reaction with a chemical mechanism different from that of its parent phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This finding demonstrates the possibility of an alternative route for the chemical reaction catalyzed by classical nonphosphorylating aldehyde dehydrogenases.     

Role of the S1' subsite glutamine 215 in activity and specificity of stromelysin-3 by site-directed mutagenesis.

The influence of Gln215 in stromelysin-3 (MMP-11), a residue located in the S1' subsite, was determined by producing three single mutants of this position. As compared to wild-type stromelysin-3, the kinetic parameters K(M) and k(cat) for the degradation of the fluorogenic substrate Dns-Pro-Leu-Ala-Leu-Trp-Ala-Arg-NH(2) (Dns-Leu) by these mutants indicated that the Gln/Leu substitution led to a 4-fold decrease in catalytic efficiency, whereas the mutations Gln/Tyr and Gln/Arg increased this parameter by a factor 10. The cleavage of alpha1-protease inhibitor (alpha1-PI), a natural substrate of stromelysin-3, by these mutants was also determined. Their relative activities for the degradation of alpha1-PI correspond to those observed with the synthetic substrate Dns-Leu. The catalytic efficiency of wild-type stromelysin-3 and its mutants to cleave the P1' analogue of Dns-Leu, containing the unusual amino acid Cys(OMeBn) (Dns-Cys(OMeBn)), was also determined. The values of the specificity factor, calculated as the ratio (k(cat)/K(M))Dns-Cys(OMeBn))/(k(cat)/K(M))Dns-Leu, were observed to vary from 26 for the wild-type stromelysin-3 to 120 for the Gln/Leu mutant and 25 for the Gln/Arg mutant. The Gln/Tyr mutant did not cleave the substrate when its P1' position is substituted by the unusual amino acid Cys(OMeBn). Altogether these observations established that both the catalytic activity and the specificity of stromelysin-3 are dependent on the nature of the residue in position 215. Finally, the cleavage efficiency of the Dns substrates by three representative matrixins, namely, MMP-14 (215 = Leu), MMP-1 (215 = Arg), and MMP-7 (215 = Tyr), was determined. Interestingly, the trends observed for these enzymes were similar to those established for the three mutants of stromelysin-3, pointing out the influence of position 215 toward the selectivity in this family of enzymes.     

Mutational evidence of transition state stabilization by serine 88 in Escherichia coli type I signal peptidase

Type I signal peptidase (SPase I) catalyzes the hydrolytic cleavage of the N-terminal signal peptide from translocated preproteins. SPase I belongs to a novel class of Ser proteases that utilize a Ser/Lys dyad catalytic mechanism instead of the classical Ser/His/Asp triad found in most Ser proteases. Recent X-ray crystallographic studies indicate that the backbone amide nitrogen of the catalytic Ser 90 and the hydroxyl side chain of Ser 88 might participate as H-bond donors in the transition-state oxyanion hole. In this work, contribution of the side-chain Ser 88 in Escherichia coli SPase I to the stabilization of the transition state was investigated through in vivo and in vitro characterizations of Ala-, Cys-, and Thr-substituted mutants. The S88T mutant maintains near-wild-type activity with the substrate pro-OmpA nuclease A. In contrast, substitution with Cys at position 88 results in more than a 740-fold reduction in activity (k(cat)) whereas S88A retains much less activity (>2440-fold decrease). Measurements of the kinetic constants of the individual mutant enzymes indicate that these decreases in activity are attributed mainly to decreases in k(cat) while effects on K(m) are minimal. Thermal inactivation and CD spectroscopic analyses indicate no global conformational perturbations of the Ser 88 mutants relative to the wild-type E. coli SPase I enzyme. These results provide strong evidence for the stabilization by Ser 88 of the oxyanion intermediate during catalysis by E. coli SPase I.     

Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies

The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.     

Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S and S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (S-KHPB). These mutations improve the value of k(cat)/K(M)(S-KHPB) by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of k(cat)(S-KHPB) for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximal value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate, a nonfunctionalized hydrophobic analogue. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate aldolases. Furthermore, targeting mutations to the active site provides a marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to those of naturally occurring enzymes.     

Substrate-selective mechanisms in biocatalysis demonstrated with a versatile and efficient aldolase antibody.

A structure-activity relationship study with a series of aldol substrates shows that the mechanism of the antibody 38C2-catalyzed retrograde aldol reaction depends on the nature of the substrate With electron-deficient substrates an early deprotonation precedes the C-C bond cleavage while with electron-rich substrates the catalytic mechanism involves an initial C-C bond cleavage leading to a positively charged intermediate.     

Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli

Menaquinone is a lipid-soluble molecule that plays an essential role as an electron carrier in the respiratory chain of many bacteria. We have previously shown that its biosynthesis in Escherichia coli involves a new intermediate, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and requires an additional enzyme to convert this intermediate into (1 R,6 R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). Here, we report the identification and characterization of MenH (or YfbB), an enzyme previously proposed to catalyze a late step in menaquinone biosynthesis, as the SHCHC synthase. The synthase catalyzes a proton abstraction reaction that results in 2,5-elimination of pyruvate from SEPHCHC and the formation of SHCHC. It is an efficient enzyme ( k cat/ K M = 2.0 x 10 (7) M (-1) s (-1)) that provides a smaller transition-state stabilization than other enzymes catalyzing proton abstraction from carbon acids. Despite its lack of the proposed thioesterase activity, the SHCHC synthase is homologous to the well-characterized C-C bond hydrolase MhpC. The crystallographic structure of the Vibrio cholerae MenH protein closely resembles that of MhpC and contains a Ser-His-Asp triad typical of serine proteases. Interestingly, this triad is conserved in all MenH proteins and is essential for the SHCHC synthase activity. Mutational analysis found that the catalytic efficiency of the E. coli protein is reduced by 1.4 x 10 (3), 2.1 x 10 (5), and 9.3 x 10 (3) folds when alanine replaces serine, histidine, and aspartate of the triad, respectively. These results show that the SHCHC synthase is closely related to alpha/beta hydrolases but catalyzes a reaction mechanistically distinct from all known hydrolase reactions.     

Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate

To investigate the role of the phosphate group 3' to the scissile phosphodiester bond of the substrate in the catalytic mechanism of Escherichia coli ribonuclease HI (RNase HI), we have used modified RNA-DNA hybrid substrates carrying a phosphorothioate substitution at this position or lacking this phosphate group for the cleavage reaction. Kinetic parameters of the H124A mutant enzyme, in which His(124) was substituted with Ala, as well as those of the wild-type RNase HI, were determined. Substitution of the pro-R(p)-oxygen of the phosphate group 3' to the scissile phosphodiester bond of the substrate with sulfur reduced the k(cat) value of the wild-type RNase HI by 6.9-fold and that of the H124A mutant enzyme by only 1. 9-fold. In contrast, substitution of the pro-S(p)-oxygen of the phosphate group at this position with sulfur had little effect on the k(cat) value of the wild-type and H124A mutant enzymes. The results obtained for the substrate lacking this phosphate group were consistent with those obtained for the substrates with the phosphorothioate substitutions. In addition, a severalfold increase in the K(m) value was observed by the substitution of the pro-R(p)-oxygen of the substrate with sulfur or by the substitution of His(124) of the enzyme with Ala, suggesting that a hydrogen bond is formed between the pro-R(p)-oxygen and His(124). These results allow us to propose that the pro-R(p)-oxygen contributes to orient His(124) to the best position for the catalytic function through the formation of a hydrogen bond.     

The key role of a non-active-site residue Met148 on the catalytic efficiency of meta-cleavage product hydrolase BphD

meta-Cleavage product (MCP) hydrolases (EC 3.7.1.9) can catalyze a specific C–C bond fission during the microbial aerobic degradation of aromatics. The previous studies on structure–function relationship of MCP hydrolases mainly focus on the active site residues by site-directed mutagenesis. However, the information about the role of the non-active-site residues is still unclear. In this study, a non-active-site residue Met148 of MCP hydrolase BphD was selected as the mutagenesis site according to the sequence alignments, structure superimpose and the tunnel analysis, which underwent the saturation mutagenesis resulting 19 mutants. The catalytic efficiencies of the mutants on 6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) were all decreased compared with the wild-type one except for the M148D mutant. Especially, the M148P mutant exhibited 290-fold lower k _cat/K _m than that of the wild-type BphD. Transient kinetic analyses of M148P showed the reciprocal relaxation time corresponded to C–C bond cleavage and product release steps (9.6 s^?1) was 4.08-fold lower than BphD WT (39.2 s^?1). Tunnel cluster analysis of BphD WT, M148P and M148W demonstrated that only the bulky Trp148 could block tunnel T2 in the BphD WT, but it exhibited slight effects on the catalytic efficiency (0.94-fold of BphD WT). Therefore, product release was not the main reason for the efficiency decrease of M148P. On the other hand, molecular dynamics simulations on the BphD WT and BphD M148P in complex with HOPDA indicated that the dramatic decrease of the catalytic efficiencies of BphD M148P should be due to the unproductive binding of HOPDA. The study demonstrated the catalytic efficiency of MCP hydrolase can be engineered by modification of non-active site residue.     

Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2

Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2. These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity. Pre-steady-state studies have shown that both Kex2 and furin exhibit an initial burst of 7-amino-4-methylcoumarin release in cleavage of peptidyl methylcoumarinamide substrates that are based on physiological cleavage sites. Thus, in cleavage of such substrates, formation of the acylenzyme intermediate is fast relative to some later step (deacylation or N-terminal product release). This behavior is significant, because Kex2 also exhibits burst kinetics in cleavage of peptide bonds. k(cat) for cleavage of a tetrapeptidyl methylcoumarinamide substrate based on the physiological yeast substrate pro-alpha-factor exhibits a weak solvent isotope effect, but neither this isotope effect nor temperature dependence studies with this substrate conclusively identify the rate-limiting step for Kex2 cleavage of this substrate. We therefore developed an assay to measure deacylation directly by pulse-chase incorporation of H(2)(18)O in a rapid-quenched-flow mixer followed by mass spectrometric quantitation. The results given by this assay rule out rate-limiting product release for cleavage of this substrate by Kex2. These experiments demonstrate that cleavage of the acylenzyme ester bond, as opposed to either the initial attack on the amide bond or product release, is rate-limiting for the action of Kex2 at physiological sequences. This work demonstrates a fundamental difference in the catalytic strategy of proprotein processing enzymes and degradative subtilisins.     

Mechanistic studies of active site mutants of Thermomonospora fusca endocellulase E2.

Endocellulase E2 from the thermophilic bacterium Thermomonospora fusca is a member of glycosyl-hydrolase family 6 and is active from pH 4 to 10. Enzymes in this family hydrolyze beta-1,4-glycosidic bonds with inversion of the stereochemistry at the anomeric carbon. The X-ray crystal structures of two family 6 enzymes have been determined, and four conserved aspartic acid residues are found in or near the active sites of both. These residues have been mutated in another family 6 enzyme, Cellulomonas fimi CenA, and evidence was found for both a catalytic acid and a catalytic base. The corresponding residues in E2 (D79, D117, D156, and D265) were mutated, and the mutant genes were expressed in Streptomyces lividans. The mutant enzymes were purified and assayed for activity on three cellulosic substrates and 2, 4-dinitrophenyl-beta-D-cellobioside. Activity on phosphoric acid-swollen cellulose was measured as a function of pH for selected mutant enzymes. Binding affinities for each mutant enzyme were measured for two fluorescent ligands and cellotriose, and circular dichroism spectra were recorded. The results show that the roles of D117 and D156 are the same as those for the corresponding residues in CenA; D117 is the catalytic acid, and D156 raises the pK(a) of D117. No specific function was assigned to the CenA residue corresponding to D79, but in E2, this residue also assists in raising the pK(a) of D117 and is important for catalytic activity. The D265N mutant retained 7% of the wild-type activity, indicating that this residue is not playing the role of the catalytic base. Experiments were conducted to rule out contamination of the D265 enzymes by either wild-type E2 or an endogenous S. lividans CMCase.