Biomedical subjects

Lixia Tang

Publications and source records attributed to Lixia Tang.

9 recordsLinked to original sources

PubMed2006

Sequential kinetic resolution catalyzed by halohydrin dehalogenase.

A sequential kinetic resolution catalyzed by halohydrin dehalogenase was employed for the synthesis of two valuable enantiopure building blocks. Resolution of methyl 4-chloro-3-hydroxybutanoate methylester ((R,S)-2) with use of a Trp249Phe mutant of halohydrin dehalogenase yielded methyl 4-cyano-3-hydroxybutanoate methylester ((S)-4) with 96.8% ee (40% yield) and (S)-2 with 95.2% ee (41% yield). This reaction is carried out in aqueous solution under mild conditions and provides access to a useful statin side-chain building block.

Catalysis↗

PubMed2006

The X-ray structure of the haloalcohol dehalogenase HheA from Arthrobacter sp. strain AD2: insight into enantioselectivity and halide binding in the haloalcohol dehalogenase family.

Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheA(AD2) from Arthrobacter sp. strain AD2 at 2.0-A resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 shows that HheA(AD2) has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheA(AD2) can bind both enantiomers of the haloalcohol substrate 1-p-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheA(AD2). Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheA(AD2), in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.

Amino Acid Sequence↗

PubMed2005

Structural basis for the enantioselectivity of an epoxide ring opening reaction catalyzed by halo alcohol dehalogenase HheC.

Halo alcohol dehalogenase HheC catalyzes the highly enantioselective dehalogenation of vicinal halo alcohols to epoxides, as well as the reverse reaction, the enantioselective and beta-regioselective nucleophilic ring opening of epoxides by pseudo-halides such as azide and cyanide. To investigate this latter reaction, we determined X-ray structures of complexes of HheC with the favored and unfavored enantiomers of para-nitrostyrene oxide. The aromatic parts of the two enantiomers bind in a very similar way, but the epoxide ring of the unfavored (S)-enantiomer binds in a nonproductive inverted manner, with the epoxide oxygen and Cbeta atom positions interchanged with respect to those of the favored (R)-enantiomer. The calculated difference in relative Gibbs binding energy is in agreement with the observed loss of a single hydrogen bond in the S bound state with respect to the R bound state. Our results indicate that it is the nonproductive binding of the unfavored (S)-enantiomer, rather than the difference in affinity for the two enantiomers, that allows HheC to catalyze the azide-mediated ring opening of para-nitrostyrene oxide with high enantioselectivity. This work represents a rare opportunity to explain the enantioselectivity of an enzymatic reaction by comparison of crystallographic data on the binding of both the favored and unfavored enantiomers.

Alcohols↗

PubMed2005

Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site.

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.

Asparagine↗

PubMed2003

Steady-state kinetics and tryptophan fluorescence properties of halohydrin dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the active site and halide-induced conformational change.

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 is a homotetrameric protein containing four tryptophan residues per subunit. The fluorescence properties of the enzyme are strongly influenced by halide binding. To examine the role of the tryptophans (W139, W192, W238, and W249) in halide binding and catalysis, they were individually mutated to a phenylalanine. All mutations, except for W238F, influenced the enzymatic properties. Mutating W192 to phenylalanine inactivated the enzyme and led to dissociation into dimers and monomers. In the structure of HheC, residue W139 and residue W249 from the opposite subunit are close to the active site of the enzyme. Substitution of W139 mainly affected K(m) values with all tested substrates and reduced the enantiopreference for p-nitro-2-bromo-1-phenylethanol. Replacing W249 increased both k(cat) and K(m) values with all tested substrates except for the (S)-enantiomer of p-nitro-2-bromo-1-phenylethanol, for which k(cat) was 3-fold decreased, resulting in a 6-fold increase of the enantioselectivity. Fluorescence measurements revealed that in the ligand-free state the intrinsic protein fluorescence of mutant W139F is higher than that of the wild-type enzyme, while the fluorescence intensity of mutants W238F and W249F was lower. The fluorescence intensities of the W238F and W249F enzymes were increased when they were unfolded or when bromide was added, whereas the fluorescence of mutant W139F was not increased by unfolding or addition of bromide. These results demonstrate that the fluorescence of residues W238 and W249 is partially quenched in the folded ligand-free state, and that W139 is completely quenched and acts as an energy acceptor for the other tryptophan residues as well. Changes of the maximum fluorescence emission wavelength of the HheC variants and the results of acrylamide quenching experiments confirmed that bromide binding induces a local conformational change around the active site, resulting in residue W139 and the quencher group being separated.

Acrylamide↗

PubMed2003

Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter.

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.

Binding Sites↗

PubMed2002

[Relationship between cyclooxygenase-2 protein expression, prostaglandins levels and biologic behavior in ovarian carcinoma tissue].

OBJECTIVE: To study the relationship between cyclooxygenase-2 (COX-2) protein expression, prostaglandins levels assay and ovarian carcinoma biologic behavior in ovarian carcinoma tissue. METHODS: The levels of COX-2 protein, prostaglandin (PG) E(2), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2) in 54 biopsy specimens from patients with ovarian serous tumor which included three groups: 33 samples of ovarian serous carcinomas; 10 samples of borderline ovarian serous tumors and 11 samples of benign ovarian serous tumors and 10 samples of normal ovarian tissues were detected by western blot analysis and radioimmunoassay to investigate their clinical significance. RESULTS: (1) The levels of COX-2 protein expression (82%, 27/33) and relative quantity (20.08 +/- 3.53) in ovarian serous tumor tissues were statistically higher than those in benign ovarian serous tumor tissues and in normal ovary tissues [0 and (15.04 +/- 0.12), 0 and (15.33 +/- 0.60), P < 0.05]. The level of COX-2 protein expression in borderline ovarian serous tumor tissues (90%, 9/10) and relative quantity (20.61 +/- 3.03) were statistically higher than those in benign ovarian serous tumor and normal ovary tissues (P < 0.05). The levels of COX-2 protein expression and relative quantity were found no significant differences in different clinical stages (I to II and III to IV), different histological grades, with or without ascites and lymphatic metastasis. (2) The levels of PGE(2), 6-keto-PGF(1 alpha) and TXB(2) in ovarian serous carcinoma tissues were statistically higher than in borderline ovarian serous tumor, benign ovarian serous tumor or normal ovarian tissues (P < 0.05). No significant differences of the levels were found among borderline tissues, benign tissues or normal ovarian tissues (P > 0.05). PGE(2), 6-keto-PGF(1 alpha) and TXB(2) were found no significant differences in different clinical stages (I to II and III to IV), different histological grades, with or without ascites and lymphatic metastasis. (3) COX-2 expression was correlated with PGE(2), 6-keto-PGF(1 alpha) and TXB(2) (P < 0.01). CONCLUSIONS: (1) Our data suggest that COX-2 overexpression leads to increased PGE(2), 6-keto-PGF(1 alpha) and TXB(2) biosynthesis, which may be mechanisms underlying the contribution of COX-2 to the development of ovarian serous carcinoma. (2) PGE(2), 6-keto-PGF(1 alpha) and TXB(2) may be helpful parameters of diagnosis and differentiate diagnosis in ovarian serous carcinoma.

6-Ketoprostaglandin F1 alpha↗

PubMed2002

[Expression of hepatitis C virus envelope proteins with a recombinant baculovirus expression system].

OBJECTIVE: To acquire stable expression of envelope proteins of hepatitis C virus in insect host cells and use the expressed envelope proteins for detecting the serums of patients with hepatitis C. METHODS: The envelope gene of HCV H strain was amplified by PCR and inserted in baculovirus vector BacPAK8, and then recombined with linear BacPAK6 DNA in insect cells. The recombinant baculoviruses were selected by the plaque assay. The insect cells were infected by the recombinant baculoviruses that contained the target gene produced E1, E2 proteins, which were characterized with the immunoblot assay and the immunofluorescence and were used to determine 35 serum samples of patients with hepatitis C. RESULTS: The expressed E1, E2 proteins showed that the relative molecular mass of E1 is about 21 x 10(3) and 33 x 10(3), and that of E2 is about 60 x 10(3). Detection of immunofluorescence indicated that E1, E2 proteins are localized in the cytoplasm of the infected cells. Four of the 35 serums responded to expressed E1; one of them was found to recognize E2 protein. Three of 9 serums which were HCV RNA positive by PCR testing got united to E1, E2. CONCLUSION: The HCV envelope protein can be expressed stably in the insect cells. Expressed E proteins could be used in the serologic analysis of the patients' serums.

Animals↗

PubMed2001

Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1.

Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop vapour-diffusion experiments against 50-70% saturated ammonium sulfate solution at pH 6.5-7.3. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 104.5, c = 121.4 A, and contain two monomers in the asymmetric unit. The crystals diffract to 3.0 A resolution with X-rays from a Cu Kalpha rotating-anode generator.

Crystallization↗