Structural and functional analysis of the D-alanyl carrier protein ligase DltA from Staphylococcus aureus Mu50

Abstract

D-Alanylation of the teichoic acids of the Gram-positive bacterial cell wall plays crucial roles in bacterial physiology and virulence. Deprivation of D-alanine from the teichoic acids of Staphylococcus aureus impairs biofilm and colony formation, induces autolysis and ultimately renders methicillin-resistant S. aureus highly susceptible to antimicrobial agents and host defense peptides. Hence, the D-alanylation pathway has emerged as a promising antibacterial target against drug-resistant S. aureus. D-Alanylation of teichoic acids is mediated via the action of four proteins encoded by the dlt operon, DltABCD, all four of which are essential for the process. In order to develop novel antimicrobial agents against S. aureus, the D-alanyl carrier protein ligase DltA, which is the first protein in the D-alanylation pathway, was focused on. Here, the crystal structure of DltA from the methicillin-resistant S. aureus strain Mu50 is presented, which reveals the unique molecular details of the catalytic center and the role of the P-loop. Kinetic analysis shows that the enantioselectivity of S. aureus DltA is much higher than that of DltA from other species. In the presence of DltC, the enzymatic activity of DltA is increased by an order of magnitude, suggesting a new exploitable binding pocket. This discovery may pave the way for a new generation of treatments for drug-resistant S. aureus.

Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains

“…Despite AlmE displaying promiscuity for D-Ala activation, this residue was never successfully transferred to the downstream T domain, and crystals could not be obtained with the replacement of Gly with D-Ala. 61 Indeed, DltA displays an increased affinity for its native substrate D-Ala, and concomitant decreased affinity for L-Ala, in the presence of coenzyme A (CoA) as a Ppant mimic or its cognate Ppant-modied T domain. 25,75 2…”

“…72 This is reminiscent of the glycylation of lipopolysaccharides described above, and moreover, AlmE shows both active site similarity to DltA and relaxed substrate selectivity towards d -Ala. 61 DltA has been crystallized several times from various organisms, and separate investigations of the active site binding pocket broadly corroborate each other. 20,25,73–76 The first of these reports identified several residues important for coordination of both the α-amino group (D197, G295 and V301) and the d -methyl group (L198 and C269) in the Bacillus cereus -derived enzyme (Fig. 7A).…”

“…7B ), which recognizes the α-amino group with residues D197, T297 and V301 and the methyl side chain with residues L198, M200 and C268 (numbering from the B. cereus -derived structure). 75 In AlmE, two key residues were thought to allow for Gly-specific activation (L248 and C316), with L248 restricting the access of D-substrates (corresponding to L198 in DltA). The conformation of L248 was found to be influenced by interactions with surrounding residues.…”