title: 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.
abstract: 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 3.2A 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.
authors.affiliation: Department of Biochemistry, 2205 Tech Drive, Northwestern University, Evanston, IL 60208-3500, USA. f-neuhaus@northwestern.edu
authors.forename: Francis C
authors.initials: FC
authors.lastname: Neuhaus
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019729:Carbon-Oxygen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003198:Computer Simulation; D024881:Drug Resistance, Bacterial; D019344:Lactic Acid / Q000737:chemistry / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology*; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D013379:Substrate Specificity; D020713:Vancomycin Resistance
chemical_list: D001426:Bacterial Proteins; C071437:VanA ligase, Bacteria; D019344:Lactic Acid; D001120:Arginine; D019729:Carbon-Oxygen Ligases; D000409:Alanine