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.
Tang
Lixia
L
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Torres Pazmiño
Daniel E
DE
Fraaije
Marco W
MW
de Jong
René M
RM
Dijkstra
Bauke W
BW
Janssen
Dick B
DB
D001216:Asparagine / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001965:Bromides / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002712:Chlorides / Q000737:chemistry* / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012231:Rhizobium / Q000201:enzymology / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D001426:Bacterial Proteins; D001965:Bromides; D002712:Chlorides; D008024:Ligands; D014443:Tyrosine; D010649:Phenylalanine; D001216:Asparagine; D014364:Tryptophan; D006867:Hydrolases; C430848:halohydrin dehalogenase