Role of the omega loop in specificity determination in subsite 2 of the d-alanine:D-alanine (d-lactate) ligase from Leuconostoc mesenteroides: A molecular docking study

The synthesis of d-ala-d-lactate in Leuconostoc mesenteroides is catalyzed by d-alanine:d-alanine (d-lactate) ligase (ADP). The ability to assemble this depsipeptide as well as d-ala-d-ala provides a mechanism for the organism's intrinsic resistance to vancomycin. Mutation of Phe261 to Tyr261 in the Ω-loop of this ligase showed a complete loss of the ability to make d-ala-d-lactate (Park and Walsh, J. Biol. Chem. 272 (1997) 9210–9214). Phe261 is a key specificity determinant in the α-helical cap of the Ω-loop when folded into the closed conformation. A molecular docking study of the closed ligase using AutoDock 4.2 defines additional specificity constraints promoted by the Ω-loop capping the catalytic center.Attaining productive orientations of d-lactate with favorable ligation chemistry requires the flexibilities of Phe261 and Arg301 in the docking protocol. These are in addition to the optimization of van der Waals contacts with Lys260, Met326, and Ser327. The location of Phe261 and Lys260 in the α-helical cap of the Ω-loop over subsite 2 is an essential part of the folding process ensuring depsipeptide formation in the hydrophobic environment of the catalytic center. The importance of the F261Y mutation suggests that the hydroxyl of Tyr261 plays an instrumental role in determining non-productive docking orientations of d-lactate. Two of these are presented: (A) d-lactate-OH as an H-bond donor to the Tyr261-OH; (B) d-lactate as an H-bond donor to the phosphoryl of the intermediate d-alanyl phosphate, and the d-lactate-COO− as an H-bond acceptor for the Tyr261-OH. Neither orientation, A or B, show the bifurcated H-bonding with Arg301 recently proposed for the activation of the nucleophilic d-lactate for d-ala-d-lactate formation. Insights into the role of the Ω-loop and its K(F/Y) signature provide additional background for inhibitor design targeted to subsite 2 of the d-alanine:d-alanine (d-X) ligases.

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► A molecular docking study of subsite 2 in the complete d-alanine:d-alanine (d-lactate) ligase from Leuconostoc mesenteroides defines specificity constraints determined by the Ω-loop capping the catalytic center.
► Favorable poses of d-lactate require the flexibilities of Phe261 and Arg301.
► Phe261 and Lys260 in the cap over subsite 2 are essential for d-ala-d-lactate poses.
► Mutation of Phe261 to Tyr261 results in non-productive poses of d-lactate.