Role of Arg301 in substrate orientation and catalysis in subsite 2 of D-alanine: D-alanine (D-lactate) ligase from Leuconostoc mesenteroides: A molecular docking study
D-Alanine:D-alanine (D-lactate) ligase (ADP) from Leuconostoc mesenteroides synthesizes the depsipeptide, D-alanyl-D-lactate, in addition to D-alanyl-D-alanine, when D-alanine and D-lactate are incubated simultaneously. The depsipeptide is responsible for the intrinsic resistance of this organism to vancomycin. The orientations of D-lactate and D-alanine in subsite 2 of the ligase that result in both nucleophile generation and subsequent attack on the electrophilic center of D-alanyl phosphate in subsite 1 are not known. A molecular docking study using AutoDock 4 suggests a role for Arg301 in determining these orientations of acceptor substrate in subsite 2 for both nucleophile generation and subsequent attack on the phosphate intermediate. With D-lactate a bifurcated H-bond from Arg301 to the R-OH of D-lactate may account for its orientation and nucleophile activation. This orientation is observed when the guanidino side chain of this residue is flexible. D-Alanine adopts an orientation that utilizes H-bonding to water 2882 and the D-alanyl phosphate in subsite 1. Both of these orientations provide mechanisms of deprotonation and place the nucleophile within 3.2 angstrom of the electrophilic carbonyl of the D-alanyl phosphate intermediate for formation of the transition state. These results suggest that Arg301 has a dual function in a sequential reaction mechanism, i.e. substrate orientation in subsite 2 as well as stabilization of the transition state. In addition, these docking studies provide insights for inhibitor design targeted to this subsite of the ligase. (C) 2010 Elsevier Inc. All rights reserved.
d-lactate molecular docking d-alanine:d-alanine (d-lactate) ligase (adp) autodock 4 vancomycin resistance