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

Abstract

The catalytic site of haloalkane dehalogenase DhlA is buried more than 10 A from the protein surface. While potential access channels to this site have been reported, the precise mechanism of substrate import and product export is still unconfirmed. We used computational methods to examine surface pockets and their putative roles in ligand access to and from the catalytic site. Computational solvent mapping moves small organic molecule as probes over the protein surface in order to identify energetically favorable sites, that is, regions that tend to bind a variety of molecules. The mapping of three DhlA structures identifies seven such regions, some of which have been previously suggested to be involved in the binding and the import/export of substrates or products. These sites are the active site, the putative entrance of the channel leading to the active site, two pockets that bind Br- ions, a pocket in the slot region, and two additional sites between the main domain and the cap of DhlA. We also performed mapping and free energy 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 program developed for finding routes from protein clefts and cavities to the surface. Solvent mapping precisely reproduced all three Br- binding sites identified by protein crystallography and the openings to four channels found by CAVER. The analyses suggest that (i) the active site has the highest affinity for the substrate molecule, (ii) the substrate initially binds at the entrance of the main tunnel, (iii) the site Br2, close to the entrance, is likely to serve as an intermediate binding site in product export, (iv) the site Br3, induced in the 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 contain significantly larger and more solvent accessible binding sites than DhlA. The mapping of DhaA and LinB places the majority of probes in the active site, but most of the other six regions consistently identified in DhlA were not observed, suggesting that the more open active site eliminates the need for intermediate binding sites for the collision complex seen in DhlA.

Pathways and Mechanisms for Product Release in the Engineered Haloalkane Dehalogenases Explored Using Classical and Random Acceleration Molecular Dynamics Simulations

“…Many globular enzymes possess active sites buried in the protein core, and there is growing evidence that the access of substrates to the active site or release of products can be a determinant of their catalytic activity [1][2][3][4][5][6][7][8][9][10][11][12][13] and substrate specificity. [1][2][3][5][6][7][8]10,[13][14][15][16][17][18][19][20][21] Mechanisms of ligand exchange between buried active sites and bulk solvent and the effects of mutations on the exchange process are often less well understood than the mechanisms of chemical reactions taking place in the active sites.…”

“…[1][2][3][5][6][7][8]10,[13][14][15][16][17][18][19][20][21] Mechanisms of ligand exchange between buried active sites and bulk solvent and the effects of mutations on the exchange process are often less well understood than the mechanisms of chemical reactions taking place in the active sites.…”

Biochemical Characterization of Haloalkane Dehalogenases DrbA and DmbC, Representatives of a Novel Subfamily

“…acting on halogenated aliphatic hydrocarbons catalyze carbon-halogen bond cleavage, leading to an alcohol, a halide ion, and a proton as the reaction products (7). Haloalkane dehalogenases originating from various bacterial strains have potential for application in bioremediation technologies (4,6,22), construction of biosensors (2), decontamination of warfare agents (17), and synthesis of optically pure compounds (19). Recent evolutionary study of haloalkane dehalogenase sequences revealed the existence of three subfamilies, denoted HLD-I, HLD-II, and HLD-III (3).…”

Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities

“…It is assumed that S 2 and S 3 play important roles in the exchange of modulators between the active site and the reaction medium when the direct channel to the active site is not easily accessible [49]. In fact, when tropolone is in MTPa, MTPb and MTPc at the end of the channels S 2 and S 3 , respectively, are the sites that show higher affinity for binding [50]. Possible energetically favorable channels in 2Y9X; A) the identified channels in snapshot 4 of 10ns MD simulation are depicted as color spheres: channel S 1 (green), S 2 (blue) and S 3 (purple).…”