The UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis utilizes Mn2+ cluster for catalysis.

Journal Article

In Escherichia coli and the majority of β- and γ-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn(2+). EPR studies reveal the presence of a Mn(2+) cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn(2+) cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.

Full Text

Duke Authors

Cited Authors

  • Young, HE; Donohue, MP; Smirnova, TI; Smirnov, AI; Zhou, P

Published Date

  • September 20, 2013

Published In

Volume / Issue

  • 288 / 38

Start / End Page

  • 26987 - 27001

PubMed ID

  • 23897835

Electronic International Standard Serial Number (EISSN)

  • 1083-351X

Digital Object Identifier (DOI)

  • 10.1074/jbc.M113.497636

Language

  • eng

Conference Location

  • United States