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Enzymatic and structural studies of UDP‐2,3‐diacylglucosamine hydrolysis in lipid A biosynthesis
Author(s) -
Young Hayley E.,
Metzger Louis E.,
Lee John K.,
Stroud Robert M.,
Zhou Pei,
Raetz Christian R. H.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.791.2
Subject(s) - lipid a , lipid ii , bacterial outer membrane , biochemistry , enzyme , biosynthesis , chemistry , escherichia coli , biology , bacteria , stereochemistry , gene , genetics
The outer‐leaflet of the outer membrane of Gram‐negative bacteria is composed of lipopolysaccharide (LPS), which is attached to the membrane via a hexa‐acylated saccharolipid called lipid A. The fourth step of the lipid A biosynthetic pathway, involving the conversion of UDP‐2,3‐diacylglucosamine (UDP‐DAGn) to 2,3‐diacylglucosamine 1‐phosphate (lipid X) and UMP, can be carried out by either LpxH or LpxI. These proteins do not share sequence homology, are never found in the same organism, and use different chemical mechanisms to catalyze the formation of lipid X. We have cloned the H. influenzae ortholog of LpxH (HiLpxH) in E. coli, purified it to 95% homogeneity, and determined its kinetic parameters and required cofactors in vitro . Mass spectrometry reveals that lipid X co‐purifies with HiLpxH. Interestingly, this property was also observed in the purification of C. crescentus LpxI, suggesting that the enzymes may share a product‐binding site. To further investigate this possibility, we have pursued structural studies of LpxH using a thermophilic ortholog from M. capsulatus (McLpxH). McLpxH has been overexpressed in E. coli and purified it to 95% homogeneity. Overall, this work sets the scene for further structural and mechanistic comparisons of LpxH and LpxI. This work is supported by NIH grant GM051310 to C.R.H.R. and Specialized Center grant of the Protein Structure Initiative U54 GM074929‐01 to R.M.S.