Holmium:YAG laser ablation combined intraurethral fluorouracil perfusion as treatment option for intraurethral Condyloma acuminata in men.

AIM: Intraurethral condylomata acuminata (CA) is caused by human papilloma virus (HPV) infection which is transmitted by close physical and sexual contact. CA is often difficult to cure. There is limited research on the treatment of the patients with intraurethral CA. Here, we have reviewed our experiences on the treatment of intraurethral condylomatous with Holmium:YAG Laser ablation. A new and convenient mean of administering fluorouracil and lidocaine for the treatment of intraurethral condyloma acuminata is discussed. This study aimed to evaluate the experience and efficacy of Holmium:YAG Laser ablation with ureteroscopy and local administration of fluorouracil in the treatment of patients with intraurethral CA. The effects were investigated based on the rate of cure and relapse and the incidence of complications. METHODS: The study included patients with intraurethral condylomatous who had undergone Holmium:YAG Laser ablation and intraurethral perfusion of fluorouracil. From May 2005 to October 2008, 25 patients (mean age 31.3 years, 19-63 years) with cystourethroscopy confirmed extensive lesions at the anterior urethra and biopsy of the lesions was compatible with condyloma acuminata. They all underwent Holmium:YAG Laser ablation with a transurethral Wolf 8/9.8 Fr rigid ureteroscope. And a week later, the patients initially accepted intraurethral installation of the mixture containing 1% fluorouracil and 1% tetracaine hydrochloride gel (lubricating jelly) in a volume of 20 mL. This mixture was given intraurethrally once weekly, and tip of the penis was clamped immediately to close the urethral meatus after administration by using an occlusive penile clamp and was retained for 20 minutes. Six treatments were given initially and after six weeks of rest, another cycle of six weekly treatments was given. RESULTS: Ureteroscopic Holmium laser ablation was successfully performed in all patients with multifocal intraurethral CA. Mean CA warts body size was 3 mm (2-8) in diameter. Mean operative time was 22.8 minutes (range 13-41). No major intraoperative complications occurred. Intraurethral installation was well tolerated, although six patients complained occasional urethral pain while urinating. Three relapses in a 2-5 weeks of follow-up underwent repeat holmium laser ablation and installation of the fluorouracil mixture. In an average of six months of follow-up, the patients have no ureteral stricture or relapse of the CA. CONCLUSION: The results of this study suggest that holmium:YAG laser ablation of the intraurethral CA combined with intraurethral perfusion of 5-fluorouracil and tetracaine hydrochloride gel mixture is an effective and safer therapy with a lower relapse rate for treatment of intraurethral CA.

GSTP1 is a hub gene for gene-air pollution interactions on childhood asthma.

There is growing evidence that multiple genes and air pollutants are associated with asthma. By identifying the effect of air pollution on the general population, the effects of air pollution on childhood asthma can be better understood. We conducted the Taiwan Children Health Study (TCHS) to investigate the influence of gene-air pollution interactions on childhood asthma. Complete monitoring data for the ambient air pollutants were collected from Taiwan Environmental Protection Agency air monitoring stations. Our results show a significant two-way gene-air pollution interaction between glutathione S-transferase P (GSTP1) and PM10 on the risk of childhood asthma. Interactions between GSTP1 and different types of air pollutants have a higher information gain than other gene-air pollutant combinations. Our study suggests that interaction between GSTP1 and PM10 is the most influential gene-air pollution interaction model on childhood asthma. The different types of air pollution combined with the GSTP1 gene may alter the susceptibility to childhood asthma. It implies that GSTP1 is an important hub gene in the anti-oxidative pathway that buffers the harmful effects of air pollution.

Targeting ß-tubulin:CCT-ß complexes incurs Hsp90- and VCP-related protein degradation and induces ER stress-associated apoptosis by triggering capacitative Ca2+ entry, mitochondrial perturbation and caspase overactivation.

We have previously demonstrated that interrupting the protein-protein interaction (PPI) of ß-tubulin:chaperonin-containing TCP-1ß (CCT-ß) induces the selective killing of multidrug-resistant cancer cells due to CCT-ß overexpression. However, the molecular mechanism has not yet been identified. In this study, we found that CCT-ß interacts with a myriad of intracellular proteins involved in the cellular functions of the endoplasmic reticulum (ER), mitochondria, cytoskeleton, proteasome and apoptosome. Our data show that the targeted cells activate both the heat-shock protein 90 (Hsp90)-associated protein ubiquitination/degradation pathway to eliminate misfolded proteins in the cytoplasm and the valosin-containing protein (VCP)-centered ER-associated protein degradation pathway to reduce the excessive levels of unfolded polypeptides from the ER, thereby mitigating ER stress, at the onset of ß-tubulin:CCT-ß complex disruption. Once ER stress is expanded, ER stress-associated apoptotic signaling is enforced, as exhibited by cellular vacuolization and intracellular Ca2+ release. Furthermore, the elevated intracellular Ca2+ levels resulting from capacitative Ca2+ entry augments apoptotic signaling by provoking mitochondrial perturbation and caspase overactivation in the targeted cells. These findings not only provide a detailed picture of the apoptotic signaling cascades evoked by targeting the ß-tubulin:CCT-ß complex but also demonstrate a strategy to combat malignancies with chemoresistance to Hsp90- and VCP-related anticancer agents.

Acute femoral neuropathy following renal transplantation: a retrospective, multicenter study in China.

BACKGROUND: We investigated the relationship between the mode and duration of iliac artery anastomosis and acute femoral neuropathy (AFN). METHODS: A retrospective analysis was performed for 83 AFN cases from 6 transplantation centers in China. The incidence and nature of dysfunction of AFN were classified based upon the duration of iliac arterial anastomosis. No prisoners were used, and no organs from prisoners were used to obtain the data. RESULTS: The incidence of AFN was 3.6% (53/1,449) in internal iliac anastomosis (group 1), 3.1% (11/346) in external iliac anastomosis (group 2) (P > .05 vs. group 1), and was 54.2% (19/35) in internal iliac ligation with external iliac anastomosis (group 3 P < .01 vs. groups 1 and 2). In group 1, the duration of the arterial anastomosis was 20 minutes in 52 cases (98.1%). In group 2, the duration of arterial anastomosis was 20 minutes in 10 cases (91%). In group 3, the duration of the arterial anastomosis was >20 minutes in all cases; 20 cases showed injury to the iliolumbar or deep iliac circumflex artery. CONCLUSION: The incidence of AFN was associated with the selection of iliac arteries, the duration of the arterial anastomosis, and an injury to the iliolumbar or deep iliac circumflex artery.

Substrate channeling and domain-domain interactions in bifunctional thymidylate synthase-dihydrofolate reductase.

The thymidylate synthase (TS) and dihydrofolate reductase (DHFR) enzymes are found on a single polypeptide chain in several species of protozoa such as the parasitic Leishmania major. Earlier studies with the bifunctional TS-DHFR enzyme from L. major have suggested that this enzyme exhibits a phenomenon known as substrate channeling [Meek, T. D., et al. (1985) Biochemistry 24, 678-686]. This is a process by which a metabolite or intermediate is directly transferred from one enzyme active site to the next without being released free into solution. The crystal structure for the bifunctional TS-DHFR enzyme from L. major was recently solved, and it was shown that the TS active site was located 40 A from the DHFR active site [Knighton, D. R., et al. (1994) Nat. Struct. Biol. 1, 186-194]. On the basis of the crystal structure, a novel mechanism has been proposed for the channeling of the intermediate, dihydrofolate, from the TS active site to the DHFR active site [Knighton, D. R., et al. (1994) Nat. Struct. Biol. 1, 186-194]. They suggest that the dihydrofolate is transferred via an "electrostatic" channel on the protein surface which connects the two active sites. In this report, we describe the use of a rapid transient kinetic analysis in examining the kinetics of substrate channeling as well as domain-domain interactions in the bifunctional TS-DHFR from L. major.

Kinetic reaction scheme for the dihydrofolate reductase domain of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major.

In several species of protozoa, the catalytic activities for the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TS) reside on a single polypeptide chain constituting a bifunctional thymidylate synthase-dihydrofolate reductase enzyme. In most other species, however, these enzymes occur as monofunctional catalytic activities on separate enzymes. In this study, the kinetic reaction scheme for the dihydrofolate reductase activity from the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) isolated from the parasite Leishmania major is compared to that of the monofunctional DHFR purified from Escherichia coli. Examination using pre-steady-state kinetic methods reveals interesting differences between the bifunctional and monofunctional forms of the dihydrofolate reductase enzymes. The rate-limiting step in the kinetic pathway for the monofunctional E. coli enzyme is the release of product, tetrahydrofolate. In contrast, for the L. major bifunctional enzyme, the kinetic step which limits the steady-state turnover is a conformational change associated with the release of NADP+. A complete kinetic description for the dihydrofolate reductase reaction pathway for the bifunctional enzyme is presented.

Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate synthase catalysis.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate. We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis. The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity. Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs. Its IPP K(m) value has only minor change. The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat). The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type. These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding. The E213 residue in region V is also important in IPP binding as well as catalysis. Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A. Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs.

Evidence for nucleophilic catalysis in the aromatic substitution reaction catalyzed by (4-chlorobenzoyl)coenzyme A dehalogenase.

(4-Chlorobenzoyl)coenzyme A dehalogenase catalyzes the hydrolytic dehalogenation of (4-chlorobenzoyl)coenzyme A (4-CBA-CoA) to (4-hydroxybenzoyl)coenzyme A (4-HBA-CoA). Rapid-quench techniques were used in conjunction with [14C]-4-CBA-CoA to test for the formation of a covalent enzyme intermediate during catalysis. The rate of [14C]-4-CBA-CoA (37 microM) consumption in the presence of a 2-fold excess of dehalogenase (75 microM) was determined to proceed at k = 6.5 s-1, coincident with the formation of an enzyme intermediate containing covalently bound radiolabel. The radiolabeled enzyme reached a maximum level at 100 ms, corresponding to 27% of the starting [14C]-4-CBA-CoA, before declining. The kinetics of formation and consumption of the radiolabeled enzyme observed during turnover are consistent with its intermediacy in the overall reaction. A single turnover reaction carried out in 98% 18O-enriched water produced 4-HBA-CoA with 73-75% 16O and 27-25% 18O at the benzoyl ring C(4)-OH. In contrast, a multiple turnover reaction carried out in 93% H2(18)O produced 4-HBA-CoA labeled at the C(4)-OH with 89% 18O and 11% 16O. These results were interpreted as evidence for formation of an aryl enzyme intermediate during 4-CBA-CoA hydrolytic dechlorination in the dehalogenase active site.

Specificity of 4-chlorobenzoyl coenzyme A dehalogenase catalyzed dehalogenation of halogenated aromatics.

Steady-state and transient kinetic techniques were used to evaluate the efficiency of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) turnover catalyzed by 4-CBA-CoA dehalogenase from Pseudomonas sp. CBS-3. The kcat for a single turnover on the enzyme was found to be 2 s-1, while that for multiple turnovers was found to be 0.6 s-1. Catalysis rather than product release was judged to be rate limiting. Comparison of the rates of turnover of 4-bromobenzoyl-CoA (1.4 s-1), 4-iodobenzoyl-CoA (1.1 s-1), and 4-fluorobenzoyl-CoA (8 x 10(-6) s-1) indicated that cleavage of the carbon-halogen bond occurs in the rate-limiting transition state of the reaction. Structure-activity measurements made with 4-CBA-CoA analogs bearing electron-donating or -withdrawing substituents at C(2) or C(3) suggested the importance of steric/solvation effects on the enzymatic reaction and failed to provide insight into the nature of the reaction intermediate. The inhibition constants measured for benzoyl-CoA (72 microM), CoA (140 microM), and 4-chlorobenzoate (21 mM) compared to the Km measured for 4-CBA-CoA (4 microM) suggest the dominant role played by the CoA moiety in substrate anchoring.

Product distribution and pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate synthase reaction.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate. We investigated the kinetics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were found. In the presence of excess FPP, the intermediates (C(25)-C(50)) accumulated. Under a condition with enzyme and FPP in excess of IPP, instead of C(20)-geranylgeranyl pyrophosphate, C(20), C(25), and C(30) were the major products. The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Triton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k(cat) value matched the rate constant of each IPP condensation obtained from the enzyme single-turnover experiments. This suggested that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton. In the absence of Triton, the intermediates formed and disappeared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting. This was further supported by a burst product formation. Judging from the accumulation level of C(55), C(60), and C(65), their dissociation from the enzyme cannot be too slow and an even slower enzyme conformational change with a rate of 0.001 s(-1) might govern the UPPs reaction rate under the steady-state condition in the absence of Triton.

Isolation and characterization of the three polypeptide components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS-3.

The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp. CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli. The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps. The 4-chlorobenzoate:coenzyme A ligase was found to be a homodimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (Km = 100 microM), coenzyme A (Km = 80 microM), and 4-chlorobenzoate (Km = 9 microM) at a rate of 30 s-1 at pH 7.5 and 25 degrees C. Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3-dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active. The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a Km = 15 microM and kcat = 0.3 s-1 at pH 7.5 and 25 degrees C and to be catalytically inactive toward hydration of crotonyl-CoA, alpha-methylcrotonyl-CoA, and beta-methylcrotonyl-CoA. The 4-hydroxybenzoate-coenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a Km = 5 microM and kcat = 7 s-1 at pH 7.5 and 25 degrees C, and to also catalyze the hydrolyses of benzoyl-coenzyme A and 4-chlorobenzoate-coenzyme A. Acetyl-coenzyme A, hexanoyl-coenzyme A, and palmitoyl-coenzyme A were not hydrolyzed by the thioesterase.

Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway.

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.

Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.

Dehydrodolichyl diphosphate synthase (DDPPs) catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize long-chain dehydrodolichyl diphosphate, which serves as a precursor of glycosyl carrier in glycoprotein biosynthesis in eukaryotes. To perform kinetic and structural studies of DDPPs, we have expressed yeast DDPPs using Escherichia coli as the host cell. Thioredoxin and His tag were utilized to increase the solubility of the recombinant protein and facilitate its purification using Ni-nitrilotriacetic acid (NTA) column. The protein was overexpressed in E. coli but mostly existed in pellet in the absence of detergent. The low quantity of soluble DDPPs was purified using Ni-NTA, Mono Q anion-exchange, and size-column chromatographies. The protein in the pellet was solubilized with 7 M urea and purified using Ni-NTA under denaturing condition. The protein refolding was achieved via the stepwise dialysis to remove the denaturant in the presence of 6 mM beta-mercaptoethanol. Detergent n-octyl-beta-d-glucopyranoside and Triton X-100 increased the solubility of the DDPPs so that refolding can be performed at higher protein concentration. Alternatively, on-column refolding was carried out in a single step to obtain the active protein in large quantities. beta-Mercaptoethanol and Triton were both required in this quick refolding process. The kinetic studies indicated that the soluble and refolded DDPPs have comparable activities (k(cat) = 2 x 10(-4) s(-1)). Unlike its bacterial homologue, undecaprenyl diphosphate synthase, yeast DDPPs activity was not enhanced by Triton.

Crystallographic studies of phosphonate-based alpha-reaction transition-state analogues complexed to tryptophan synthase.

In an effort to use a structure-based approach for the design of new herbicides, the crystal structures of complexes of tryptophan synthase with a series of phosphonate enzyme inhibitors were determined at 2.3 A or higher resolution. These inhibitors were designed to mimic the transition state formed during the alpha-reaction of the enzyme and, as expected, have affinities much greater than that of the natural substrate indole-3-glycerol phosphate or its nonhydrolyzable analogue indole propanol phosphate (IPP). These inhibitors are ortho-substituted arylthioalkylphosphonate derivatives that have an sp(3)-hybridized sulfur atom, designed to mimic the putative tetrahedral transition state at the C3 atom of the indole, and lack the C2 atom to allow for higher conformational flexibility. Overall, the inhibitors bind in a fashion similar to that of IPP. Glu-49 and Phe-212 are the two active site residues whose conformation changes upon inhibitor binding. A very short hydrogen bond between a phosphonate oxygen and the Ser-235 hydroxyl oxygen may be responsible for stabilization of the enzyme-inhibitor complexes. Implications for the mechanism of catalysis as well as directions for more potent inhibitors are discussed.

Catalytic mechanism of Kdo8P synthase: transient kinetic studies and evaluation of a putative reaction intermediate.

The mechanistic pathway for the reaction catalyzed by Kdo8P synthase has been investigated, and the cyclic bisphosphate 2 has been examined as a putative reaction intermediate. Two parallel approaches were used: (1) chemical synthesis of 2 and evaluation as an alternate substrate for the enzyme and (2) transient kinetic studies using rapid chemical quench methodology to provide direct observation and characterization of putative intermediate(s) during enzyme catalysis. The putative cyclic bisphosphate intermediate 2, possessing the stereochemistry of the beta-pyranose form, was synthesized and evaluated as a substrate and as an inhibitor of Kdo8P synthase. The substrate activity was examined by monitoring the release of anomeric phosphate over time using proton-decoupled 31P NMR spectroscopy. A very similar time course for the formation of inorganic phosphate was found in each experiment and the corresponding control experiment; i.e., no enzyme-catalyzed acceleration in the anomeric phosphate hydrolysis was detected. It was found however that 2 binds to the enzyme and is a competitive inhibitor with respect to phosphoenolpyruvate binding, having a Ki value of 35 microM. In a parallel study, we have performed single-turnover rapid chemical quench experiments to examine both the forward and reverse directions to identify a putative enzyme intermediate(s). Our results clearly demonstrate that the cyclic bisphosphate intermediate 2 does not accumulate under single-enzyme turnover conditions. This observation, coupled with the results obtained through the evaluation of synthetic 2 as a substrate, strongly suggests that the Kdo8P synthase catalytic pathway does not involve the formation of 2 as a reaction intermediate. Taken together, these combined results support the original hypothesis [Hedstrom, L., and Abeles, R. H. (1988) Biochem. Biophys. Res. Commun. 157, 816-820], which suggests a reaction pathway involving an acyclic bisphosphate intermediate 1.

Leishmania major pteridine reductase 1 belongs to the short chain dehydrogenase family: stereochemical and kinetic evidence.

Pteridine reductase 1 (PTR1) is a novel broad spectrum enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. Overexpression of PTR1 confers methotrexate resistance to these protozoa, arising from the enzyme's ability to reduce dihydrofolate and its relative insensitivity to methotrexate. The kinetic mechanism and stereochemical course for the catalyzed reaction confirm PTR1's membership within the short chain dehydrogenase/reductase (SDR) family. With folate as a substrate, PTR1 catalyzes two rounds of reduction, yielding 5,6,7, 8-tetrahydrofolate and oxidizing 2 equiv of NADPH. Dihydrofolate accumulates transiently during folate reduction and is both a substrate and an inhibitor of PTR1. PTR1 transfers the pro-S hydride of NADPH to carbon 6 on the si face of dihydrofolate, producing the same stereoisomer of THF as does dihydrofolate reductase. Product inhibition and isotope partitioning studies support an ordered ternary complex mechanism, with NADPH binding first and NADP+ dissociating after the reduced pteridine. Identical kinetic mechanisms and NAD(P)H hydride chirality preferences are seen with other SDRs. An observed tritium effect upon V/K for reduction of dihydrofolate arising from isotopic substitution of the transferred hydride was suppressed at a high concentration of dihydrofolate, consistent with a steady-state ordered kinetic mechanism. Interestingly, half of the binary enzyme-NADPH complex appears to be incapable of rapid turnover. Fluorescence quenching results also indicate the existence of a nonproductive binary enzyme-dihydrofolate complex. The nonproductive complexes observed between PTR1 and its substrates are unique among members of the SDR family and may provide leads for developing antileishmanial therapeutics.

Loop closure and intersubunit communication in tryptophan synthase.

Crystal structures of wild-type tryptophan synthase alpha2beta2 complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the alpha-reaction by the aminoacrylate intermediate formed at the beta-active site. Using a flow cell, the aminoacrylate (A-A) intermediate of the beta-reaction () was generated in the crystal under steady state conditions in the presence of serine and the alpha-site inhibitor 5-fluoroindole propanol phosphate (F-IPP). A model for the conformation of the Schiff base between the aminoacrylate and the beta-subunit cofactor pyridoxal phosphate (PLP) is presented. The structure is compared with structures of the enzyme determined in the absence (TRPS) and presence (TRPSF-IPP) of F-IPP. A detailed model for binding of F-IPP to the alpha-subunit is presented. In contrast to findings by Hyde et al. [(1988) J. Biol. Chem. 263,17857-17871] and Rhee et al. [(1997) Biochemistry 36, 7664-7680], we find that the presence of an alpha-site alone ligand is sufficient for loop alphaL6 closure atop the alpha-active site. Part of this loop, alphaThr183, is important not only for positioning the catalytic alphaAsp60 but also for coordinating the concomitant ordering of loop alphaL2 upon F-IPP binding. On the basis of the three structures, a pathway for communication between the alpha- and beta-active sites has been established. The central element of this pathway is a newly defined rigid, but movable, domain that on one side interacts with the alpha-subunit via loop alphaL2 and on the other side with the beta-active site. These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase.

Evidence for electrophilic catalysis in the 4-chlorobenzoyl-CoA dehalogenase reaction: UV, Raman, and 13C-NMR spectral studies of dehalogenase complexes of benzoyl-CoA adducts.

This paper reports on the mechanism of substrate activation by the enzyme 4-chlorobenzoyl coenzyme A dehalogenase. This enzyme catalyzes the hydrolytic dehalogenation of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) to form 4-hydroxybenzoyl coenzyme A (4-HBA-CoA). The mechanism of this reaction is known to involve attack of an active site carboxylate (Asp or Glu side chain) at C(4) of the substrate benzoyl ring to form a Meisenheimer complex. Loss of chloride ion from this intermediate results in the formation of an arylated enzyme intermediate. The arylated enzyme is hydrolyzed to free enzyme plus 4-HBA-CoA by the addition of water at the acyl carbon [Yang, G., Liang, P.-H., & Dunaway-Mariano, D. (1994) Biochemistry 33, 8527]. The present studies have focused on the activation of the 4-CBA-CoA for nucleophilic attack by the active site carboxylate group. UV-visible, 13C-NMR, and Raman spectroscopic techniques were used to monitor changes in the distribution of the pi electrons of the benzoyl moiety of benzoyl-CoA adducts [substituted at C(4) with methyl (4-MeBA-CoA), methoxy (4-MeOBA-CoA), or hydroxyl (4-HBA-CoA) groups or at C(2) or C(3) with a hydroxyl group (2-HBA-CoA and 3-HBA-CoA)] resulting from the binding of these ligands to the dehalogenase active site. The UV-visible spectra measured for 4-HBA-CoA in aqueous buffer at pH 7.5 and in the dehalogenase active site revealed that a large red shift (from 292 to 373 nm) in the lambda max of the benzoyl moiety occurs upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases.

We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.

Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis.

The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the betaA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four beta-strands (betaA-betaB-betaD-betaC) and two helices (alpha2 and alpha3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C(70) polymer rather than the C(55) polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C(30) intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C(55) product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C(55) product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k(cat) values. W75A showed an 8-fold increase of the FPP K(m) value, and 22-33-fold increases in the IPP K(m) values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.