Polyamines are a ubiquitous class of polycationic small molecules that can influence gene expression by binding to nucleic acids. Reversible polyamine acetylation regulates nucleic acid binding and is required for normal cell cycle progression and proliferation. Here, we report the structures of Mycoplana ramosa acetylpolyamine amidohydrolase (APAH) complexed with a transition state analogue and a hydroxamate inhibitor, and an inactive mutant complexed with two acetylpolyamine substrates. The structure of APAH is the first of a histone deacetylase-like oligomer and reveals that an 18-residue insert in the L2 loop promotes dimerization and the formation of an 18-Å long “L”-shaped active site tunnel at the dimer interface, accessible only to narrow and flexible substrates. The importance of dimerization for polyamine deacetylase function leads to the suggestion that a comparable dimeric or double-domain histone deacetylase could catalyze polyamine deacetylation reactions in eukaryotes.
The first committed step of lipid A biosynthesis is catalyzed by UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase, a metal-dependent deacetylase also known as LpxC. Because lipid A is essential for bacterial viability, the inhibition of LpxC is an appealing therapeutic strategy for the treatment of Gram-negative bacterial infections. Here we report the 1.79 Å resolution X-ray crystal structure of LpxC from Yersinia enterocolitica (YeLpxC) complexed with the potent hydroxamate inhibitor CHIR-090. This enzyme is a nearly-identical orthologue of LpxC from Yersinia pestis (99.7% sequence identity), the pathogen that causes bubonic plague. Similar to the inhibition of LpxC from Escherichia coli, CHIR-090 inhibits YeLpxC via a two-step slow, tight-binding mechanism with an apparent K i of 0.54 ± 0.14 nM followed by conversion of the E•I to E•I* species with a rate constant of 0.11 ± 0.01 min −1 . The structure of the LpxC complex with CHIR-090 shows that the inhibitor hydroxamate group chelates the active site zinc ion, and the "tail" of the inhibitor binds in the hydrophobic tunnel in the active site. This hydrophobic tunnel is framed by a βαβ subdomain that exhibits significant conformational flexibility as it accommodates inhibitor binding. CHIR-090 displays a 27 mm zone of inhibition against Y. enterocolitica in a Kirby-Bauer antibiotic assay, which is comparable to its reported activity against other Gram-negative species including E. coli and Pseudomonas aeruginosa. This study demonstrates that the inhibition of LpxC should be explored as a potential therapeutic and/or prophylatic response to infection by weaponized Yersinia species.Septic shock is one of the leading causes of death in hospital intensive care units and results from severe hypotension and multiple organ failure associated with bacterial sepsis. Sepsis is a systemic inflammatory response to infection: specifically, macrophage activation by endotoxin, which is also known as lipopolysaccharide (LPS). Approximately 10 6 lipopolysaccharide molecules constitute the outer leaflet of the outer membrane of a Gramnegative bacterium (1). This "protective sheath" makes Gram-negative bacteria impermeable to many commercial antibiotics. † This work was supported by the National Institutes of Health (D.W.C. GM49758 and C.A.F. GM40602). S.G.G. was supported by NIGMS training grant (T32 GM007767 The first committed step of lipid A biosynthesis is catalyzed by UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase, a metal-dependent deacetylase also known as LpxC ( Figure 1A) (6-9). X-ray crystal structures have been reported for LpxC from the Gram-negative bacteria Aquifex aeolicus (AaLpxC) (10) and Pseudomonas aeruginosa (PaLpxC) (11), as well as various ligand complexes of AaLpxC (12-16). The structure of AaLpxC has also been determined in solution by NMR methods (17, 18), and the NMR structure of its complex with the potent, slow-binding hydroxamate inhibitor CHIR-090 ( Figure 1B) has been reported (19,20). These studies ha...
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