Structural basis for the sugar nucleotide and acyl-chain selectivity of Leptospira interrogans LpxA.

Leptospira interrogans, a spirochete that inhabits areas with warm, humid climates, thrives in fresh water sources contaminated by animal urine (1). L. interrogans infect humans who come in contact with the contaminated water or soils, causing Weil's disease, which damages the liver, kidneys, heart and lungs (1), and may be fatal if left untreated (2). L. interrogans is a spirochete that is able to survive outside of its host and one of the few that synthesizes lipopolysaccharide (LPS) (3, 4). Accordingly, all L. interrogans genomes encode orthologues of the Escherichia coli Lpx enzymes (3, 4), which are responsible for the assembly of the Kdo2-lipid A anchor of LPS (Fig. 1). Figure 1 Substrate selectivity of LiLpxA versus EcLpxA and the origin of UDP-GlcNAc3N Some Gram-negative bacteria, including L. interrogans, synthesize variants of lipid A in which a 2,3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N) unit replaces one or both glucosamine residues (5, 6). L. interrogans lipid A contains two such GlcN3N units (6) (Fig. 1A). The GlcN3N residues are derived from UDP-2-acetamido-3-amino-2,3-dideoxy-α-d-glucopyranose (UDP-GlcNAc3N) (Fig. 1A) (7, 8). This material is synthesized from UDP-GlcNAc by the sequential actions of the dehydrogenase GnnA and the transaminase GnnB; these proteins are not present in E. coli (Fig. 1A) (7, 8). L. interrogans LpxA (LiLpxA) transfers the R-3-hydroxylauroyl chain from R-3-hydroxylauroyl-ACP to UDP-GlcNAc3N (6, 7) (Fig. 1A). LiLpxA shows an absolute selectivity for UDP-GlcNAc3N (Fig. 1A) (7). EcLpxA acylates both UDP-GlcNAc and UDP-GlcNAc3N in vitro with equal efficiency (7) (not shown in Fig. 1A), but UDP-GlcNAc3N is not synthesized by wild-type E. coli. LiLpxA also shows a striking preference for R-3-hydroxylauroyl-ACP over other hydroxyacyl-ACPs (7) (Fig. 1A). E. coli LpxA (EcLpxA) is highly selective for R-3-hydroxymyristoyl-ACP (Fig. 1B), but it can also slowly utilize R-3-hydroxylauroyl- and R-3-hydroxypalmitoyl-ACP (7, 9, 10). Initial biochemical studies of EcLpxA suggested a mechanism in which H125 is the catalytic base (11) (Scheme 1). Recent structural studies have confirmed that the NE2 atom of H125 is within hydrogen-bonding distance of the 3-OH group of the glucosamine ring in complexes of EcLpxA with UDP-GlcNAc (12). These structures also elucidated the roles of the conserved EcLpxA residues Q73, L75, H99, H122, H144, Q161, N198, and R205 in substrate binding (Scheme 1) (12). The NH group of G143 is the proposed oxyanion hole (Scheme 1). The preference of EcLpxA for R-3-hydroxymyristoyl-ACP is explained by the positioning of G173 and H191 (12). The reaction catalyzed by EcLpxA with UDP-GlcNAc is reversible and thermodynamically unfavorable in the forward direction (11, 13). Scheme 1 Proposed catalytic mechanism of EcLpxA and roles of key residues in substrate binding. The structure of an LpxA orthologue selective for UDP-GlcNAc3N has not been investigated. Herein, we report the crystal structures of LiLpxA in the free form at 2.10 A, with bound UDP-3-N-(R-3-hydroxylauroyl)-GlcNAc3N at 2.10 A, and with bound R-3-hydroxylauroyl-methylphosphopantetheine at 2.12 A. The latter compound is an effective model substrate for LiLpxA but not EcLpxA. The space group is trigonal (P321). The asymmetric unit of LiLpxA contains three protein molecules, whereas that of EcLpxA contains only one (12, 14). However, the active site cleft of the biologically relevant LiLpxA homotrimer (which is not the same as three monomers in the asymmetric unit) is similar to that of EcLpxA (12), and its structure explains the R-3-hydroxylauroyl-ACP selectivity of LiLpxA. The altered position of the backbone carbonyl moiety of Q68 in LiLpxA provides a favorable H-bond acceptor for the NH2 moiety of UDP-GlcNAc3N but discriminates against the 3-OH group of UDP-GlcNAc. The UDP-GlcNAc3N selectivity of LiLpxA is reminiscent of the mechanism of bacterial resistance to vancomycin.