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.     

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.     

Evidence for a gem-diol reaction intermediate in bacterial C-C hydrolase enzymes BphD and MhpC from 13C NMR spectroscopy

C-C hydrolase enzymes MhpC and BphD catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid and Burkholderia xenovorans LB400 biphenyl degradation pathways and are both members of the alpha/beta-hydrolase family containing a Ser-His-Asp catalytic triad. The catalytic mechanism of this family of enzymes is thought to proceed via a gem-diol reaction intermediate, which has not been observed directly. Site-directed single mutants of BphD in which catalytic residues His-265 and Ser-112 were replaced with Ala were found to possess 10(4)-fold reduced k(cat) values, and in each case, the C-C cleavage step was shown by pre-steady-state kinetic analysis to be rate-limiting. The processing of a 6-(13)C-labeled aryl-containing substrate by these H265A or S112A mutant BphD enzymes was monitored directly by (13)C NMR spectroscopy. A new line-broadened signal was observed at 128 ppm for each enzyme, corresponding to the proposed gem-diol reaction intermediate, over a time scale of 1-24 h. A similar signal was observed upon incubation of the (13)C-labeled substrate with an H114A MhpC mutant, which is able to accept the 6-phenyl-containing substrate, on a shorter time scale. The direct observation of a gem-diol intermediate provides further evidence that supports a general base mechanism for this family of enzymes.     

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.     

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.     

Reaction of Pseudomonas fluorescens kynureninase with beta-benzoyl-L-alanine: detection of a new reaction intermediate and a change in rate-determining step

Beta-benzoyl-DL-alanine was synthesized from alpha-bromoacetophenone and diethyl acetamidomalonate. The racemic amino acid was resolved by carboxypeptidase A-catalyzed hydrolysis of the N-trifluoroacetyl derivative. Beta-benzoyl-L-alanine is a good substrate of kynureninase from Pseudomonas fluorescens, with k(cat) and k(cat)/K(m) values of 0.7 s(-1) and 8.0 x 10(4) M(-1) s(-1), respectively, compared to k(cat) = 16.0 s(-1) and k(cat)/K(m) = 6.0 x 10(5) M(-1) s(-1) for L-kynurenine. In contrast to the reaction of L-kynurenine, beta-benzoyl-L-alanine does not exhibit a significant solvent isotope effect on k(cat) ((H)k/(D)k = 0.96 +/- 0.06). The pre-steady-state kinetics of the reaction of beta-benzoyl-L-alanine were investigated by rapid scanning stopped-flow spectrophotometry. The spectra show the formation of a quinonoid intermediate, with lambda(max) = 490 nm, in the dead time of the instrument, which then decays, with k = 210 s(-1), to form a transient intermediate with lambda(max) at 348 nm. In the presence of benzaldehyde, the 348 nm intermediate decays, with k = 0.7 s(-1), to form a quasistable quinonoid species with lambda(max) = 492 nm. Previous studies demonstrated that benzaldehyde can trap an enamine intermediate formed after the C(beta)-C(gamma) bond cleavage [Phillips, R. S., Sundararaju, B., and Koushik, S. V. (1998) Biochemistry 37, 8783-8789]. Thus, the 348 nm intermediate is kinetically competent. The position of the absorption maximum and shape of the band is consistent with a PMP-ketimine intermediate. The results from chemical quenching analysis do not show a burst of benzoate and, thus, also support the formation of benzoate as the rate-determining step. These data suggest that, in contrast to L-kynurenine, for which the rate-determining step was shown to be deprotonation of the pyruvate-ketimine intermediate [Koushik, S. V., Moore, J. A., III, Sundararaju, B., and Phillips, R. S. (1998) Biochemistry 37, 1376-1382], the rate-determining step in the reaction of beta-benzoyl-L-alanine with kynureninase is C(beta)-C(gamma) bond cleavage.     

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.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.     

Identification of a serine hydrolase as a key determinant in the microbial degradation of polychlorinated biphenyls

The ability of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) of Burkholderia cepacia LB400 to hydrolyze polychlorinated biphenyl (PCB) metabolites was assessed by determining its specificity for monochlorinated HOPDAs. The relative specificities of BphD for HOPDAs bearing chlorine substituents on the phenyl moiety were 0.28, 0.38, and 1.1 for 8-Cl, 9-Cl, and 10-Cl HOPDA, respectively, versus HOPDA (100 mm phosphate, pH 7.5, 25 degrees C). In contrast, HOPDAs bearing chlorine substituents on the dienoate moiety were poor substrates for BphD, which hydrolyzed 3-Cl, 4-Cl, and 5-Cl HOPDA at relative maximal rates of 2.1 x 10(-3), 1.4 x 10(-4), and 0.36, respectively, versus HOPDA. The enzymatic transformation of 3-, 5-, 8-, 9-, and 10-Cl HOPDAs yielded stoichiometric quantities of the corresponding benzoate, indicating that BphD catalyzes the hydrolysis of these HOPDAs in the same manner as unchlorinated HOPDA. HOPDAs also underwent a nonenzymatic transformation to products that included acetophenone. In the case of 4-Cl HOPDA, this transformation proceeded via the formation of 4-OH HOPDA (t(12) = 2.8 h; 100 mm phosphate, pH 7.5, 25 degrees C). 3-Cl HOPDA (t(12) = 504 h) was almost 3 times more stable than 4-OH HOPDA. Finally, 3-Cl, 4-Cl and 4-OH HOPDAs competitively inhibited the BphD-catalyzed hydrolysis of HOPDA (K(ic) values of 0.57 +/- 0. 04, 3.6 +/- 0.2, and 0.95 +/- 0.04 microm, respectively). These results explain the accumulation of HOPDAs and chloroacetophenones in the microbial degradation of certain PCB congeners. More significantly, they indicate that in the degradation of PCB mixtures, BphD would be inhibited, thereby slowing the mineralization of all congeners. BphD is thus a key determinant in the aerobic microbial degradation of PCBs.     

Structure of 6-oxo camphor hydrolase H122A mutant bound to its natural product, (2S,4S)-alpha-campholinic acid: mutant structure suggests an atypical mode of transition state binding for a crotonase homolog

The crotonase homolog, 6-oxo camphor hydrolase (OCH), catalyzes the desymmetrization of bicyclic beta-diketones to optically active keto acids via an enzymatic retro-Claisen reaction, resulting in the cleavage of a carbon-carbon bond. We have previously reported the structure of OCH (Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744-1750), which suggested the involvement of five residues, His-45, His-122, His-145, Asp-154, and Glu-244, in catalysis. Here we report mutation studies on OCH that reveal that H145A and D154N mutants of OCH have greatly reduced values of k(cat)/K(m) derived from a very large increase in K(m) for the native substrate, 6-oxo camphor. In addition, H122A has a greatly reduced value of k(cat), and its K(m) is five times that of the wild-type. The location of the active site is confirmed by the 1.9-A structure of the H122A mutant of OCH complexed with the minor diastereoisomer of (2S,4S)-alpha-campholinic acid, the natural product of the enzyme. This shows the pendant acetate of the product hydrogen bonded to a His-145/Asp-154 dyad and the endocyclic carbonyl of the cyclopentane ring hydrogen bonded to Trp-40. The results are suggestive of a base-catalyzed mechanism of C-C bond cleavage and provide clues to the origin of prochiral selectivity by the enzyme and to the recruitment of the crotonase fold for alternate modes of transition state stabilization to those described for other crotonase superfamily members.     

Characterization of a Carbon-Carbon Hydrolase from Mycobacterium tuberculosis Involved in Cholesterol Metabolism

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5–9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4–33 times higher specificity for DSHA (k_cat/K_m = 3.3 ± 0.3 × 10⁴ m⁻¹ s⁻¹) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K_d of 51 ± 2 μm. The S114A·DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8–1.9 Åindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the α-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.     

An alternative mechanism of bioluminescence color determination in firefly luciferase

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) approximately 530 nm) to red (lambda(max) approximately 635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., and Stroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferin analogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorus plagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainly in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme, and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescence color that require only a single keto form of oxyluciferin. We report here the results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, as well as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized using bioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on an analysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternative mechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.     

DNA strand transfer catalyzed by vaccinia topoisomerase: peroxidolysis and hydroxylaminolysis of the covalent protein-DNA intermediate

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at sites containing the sequence 5'-CCCTT downward arrow. The covalently bound topoisomerase can religate the CCCTT strand to a 5'-OH-terminated polynucleotide or else transfer the strand to a non-DNA nucleophile such a water or glycerol. Here, we report that vaccinia topoisomerase also catalyzes strand transfer to hydrogen peroxide. The observed alkaline pH-dependence of peroxidolysis is consistent with enzyme-mediated attack by peroxide anion on the covalent intermediate. The reaction displays apparent first-order kinetics. From a double-reciprocal plot of k(obs) versus [H(2)O(2)] at pH 10, we determined a rate constant for peroxidolysis of 6.3 x 10(-)(3) s(-)(1). This rate is slower by a factor of 200 than the rate of topoisomerase-catalyzed strand transfer to a perfectly aligned 5'-OH DNA strand but is comparable to the rate of DNA strand transfer across a 1-nucleotide gap. Strand transfer to 2% hydrogen peroxide is 300 times faster than strand transfer to 20% glycerol and approximately 2000 times faster than topoisomerase-catalyzed hydrolysis of the covalent intermediate. Hydroxylamine is also an effective nucleophile in topoisomerase-mediated strand transfer (k(obs) = 6.4 x 10(-)(4) s(-)(1)). The rates of the peroxidolysis, hydroxylaminolysis, glycerololysis, and hydrolysis reactions catalyzed by the mutant enzyme H265A were reduced by factors of 100-700, in accordance with the 100- to 400-fold rate decrements in DNA cleavage and religation by H265A. We surmise that vaccinia topoisomerase catalyzes strand transfer to DNA and non-DNA nucleophiles via a common reaction pathway in which His-265 stabilizes the scissile phosphate in the transition state rather than acting as a general acid or base.     

Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations

The structural changes taking place in the enzyme thymidine phosphorylase (TPase, also known as PD-ECGF) that are required to achieve catalytic competence upon binding thymidine and phosphate have been simulated by means of targeted molecular dynamics (tMD). The hinge regions were characterized by structural homology comparisons with pyrimidine nucleoside phosphorylase, whose X-ray structure has been solved both in a closed and in an open form. The rearrangement of residues around the substrate that was observed during the tMD trajectory suggested that His-85 could be playing an important role in the catalytic mechanism. A quantum mechanical study of the reaction in the presence of the most relevant active site residues was then performed at the semiempirical level. The results revealed that His-85 could be involved in the protonation of the pyrimidine base at the O2 position to yield the enol tautomer of the base. To establish the role of this oxygen atom in the reaction, ground states, transition states, and final products were studied using higher level ab initio methods starting from both thymidine and 2-thiothymidine as alternative substrates. Comparison of both transition states showed that replacing the oxygen at position 2 of the pyrimidine base by sulfur should accelerate the reaction rate. Consistent with this result, 2-thiothymidine was shown to be a better substrate for TPase than the natural substrate, thymidine. For simulating the final step of the reaction, tMD simulations were used to study domain opening upon product formation considering both the enol and keto tautomers of thymine. Product release from the enzyme was easiest in the simulation that incorporated the keto tautomer of thymine, suggesting that the enol intermediate spontaneously tautomerizes back to the more energetically stable keto form. These results highlight a previously unreported role for His-85 in the catalytic mechanism of TPase and can have important implications for the design of novel TPase inhibitors.     

Electronic and nuclear magnetic resonance spectroscopic features of the 1',4'-iminopyrimidine tautomeric form of thiamin diphosphate, a novel intermediate on enzymes requiring this coenzyme

Appropriate compounds were synthesized to create models for the 1',4'-imino tautomer of the 4'-aminopyrimidine ring of thiamin diphosphate recently found to exist on the pathway of enzymatic reactions requiring this cofactor [Jordan, F., and Nemeria, N. S. (2005) Bioorg. Chem. 33, 190-215]. The N1-methyl-4-aminopyrimidinium compounds synthesized on treatment with a strong base produce the 1,4-imino tautomer whose UV spectrum indicates a maximum between 300 and 320 nm, depending on the absence or presence of a methyl group at the 4-amino nitrogen. The lambda(max) found is in the same wavelength range as the positive circular dichroism band observed on several enzymes and showed a very strong dependence on solvent dielectric constant. To help with the 15N chemical shift assignments, the model compounds were specifically labeled with 15N at the amino nitrogen atom. The chemical shift of the amino nitrogen was deshielded by N1-methylation and then dramatically further deshielded by more than 100 ppm on formation of the 1,4-iminopyrimidine tautomer. Both the UV spectroscopic values and the 15N chemical shift for the 1,4-iminopyrimidine tautomer should serve as useful guides to the assignment of enzyme-bound signals.     

Light-stable rhodopsin. II. An opsin mutant (TRP-265----Phe) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore.

In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117----Phe, Glu-122----Gln(Ala, Asp), Trp-126----Phe(Leu, Ala), Trp-265----Ala(Tyr, Phe), Tyr-268----Phe, and Ala-292----Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117----Phe, Glu-122----Gln(Ala), Trp-126----Leu(Ala), and Trp-265----Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117----Phe and Trp-126----Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126----Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122----Asp, Trp-265----Ala, Tyr-268----Phe, and Ala-292----Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265----Tyr, while the mutant Trp-265----Phe was light-insensitive. Consistent with this result, the mutant Trp-265----Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase.     

The 2-A crystal structure of 6-oxo camphor hydrolase. New structural diversity in the crotonase superfamily

6-Oxo camphor hydrolase (OCH) is an enzyme of the crotonase superfamily that catalyzes carbon-carbon bond cleavage in bicyclic beta-diketones via a retro-Claisen reaction (Grogan, G., Roberts, G. A., Bougioukou, D., Turner, N. J., and Flitsch, S. L. (2001) J. Biol. Chem. 276, 12565-12572). The native structure of OCH has been solved at 2.0-A resolution with selenomethionine multiple wave anomalous dispersion and refined to a final R(free) of 19.0. The structure of OCH consists of a dimer of trimers that resembles the "parent" enzyme of the superfamily, enoyl-CoA hydratase. In contrast to enoyl-CoA hydratase, however, two octahedrally coordinated sodium atoms are found at the 3-fold axis of the hexamer of OCH, and the C-terminal helix of OCH does not form a discrete domain. Models of the substrate, 6-oxo camphor, and a proposed enolate intermediate in the putative active site suggest possible mechanistic roles for Glu-244, Asp-154, His-122, His-45, and His-145.     

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.     

Di(1,N6-ethenoadenosine)5', 5'-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N6-ethenoadenosine)5',5'-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.