Exploring the Binding Sites of the Haloalkane Dehalogenase DhlA from Xanthobacter autotrophicus GJ10

详细信息    查看全文

• 作者：Michael Silberstein ; Jiri Damborsky ; Sandor Vajda
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：August 14, 2007
• 年：2007
• 卷：46
• 期：32
• 页码：9239 - 9249
• 全文大小：428K
• 年卷期：v.46,no.32(August 14, 2007)
• ISSN：1520-4995

文摘

The catalytic site of haloalkane dehalogenase DhlA is buried more than 10 Å from the proteinsurface. While potential access channels to this site have been reported, the precise mechanism of substrateimport and product export is still unconfirmed. We used computational methods to examine surface pocketsand their putative roles in ligand access to and from the catalytic site. Computational solvent mappingmoves small organic molecule as probes over the protein surface in order to identify energetically favorablesites, that is, regions that tend to bind a variety of molecules. The mapping of three DhlA structuresidentifies seven such regions, some of which have been previously suggested to be involved in the bindingand the import/export of substrates or products. These sites are the active site, the putative entrance of thechannel leading to the active site, two pockets that bind Br^- ions, a pocket in the slot region, and twoadditional sites between the main domain and the cap of DhlA. We also performed mapping and freeenergy analysis of the DhlA structures using the substrate, 1,2-dichloroethane, and halide ions as probes.The findings were compared to crystallographic data and to results obtained by CAVER, a programdeveloped for finding routes from protein clefts and cavities to the surface. Solvent mapping preciselyreproduced all three Br^- binding sites identified by protein crystallography and the openings to four channelsfound by CAVER. The analyses suggest that (i) the active site has the highest affinity for the substratemolecule, (ii) the substrate initially binds at the entrance of the main tunnel, (iii) the site Br2, close to theentrance, is likely to serve as an intermediate binding site in product export, (iv) the site Br3, induced inthe structure at high concentrations of Br^-, could be part of an auxiliary route for product release, and (v)three of the identified sites are likely to be entrances of water-access channels leading to the active site.For comparison, we also mapped haloalkane dehalogenases DhaA and LinB, both of which containsignificantly larger and more solvent accessible binding sites than DhlA. The mapping of DhaA andLinB places the majority of probes in the active site, but most of the other six regions consistently identifiedin DhlA were not observed, suggesting that the more open active site eliminates the need for intermediatebinding sites for the collision complex seen in DhlA.