The UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis utilizes Mn2+ cluster for catalysis.
J Biol Chem
; 288(38): 26987-27001, 2013 Sep 20.
Article
em En
| MEDLINE
| ID: mdl-23897835
ABSTRACT
In Escherichia coli and the majority of ß- 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.
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Texto completo:
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Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Pirofosfatases
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Proteínas de Bactérias
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Haemophilus influenzae
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Lipídeo A
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Manganês
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2013
Tipo de documento:
Article