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J. Biol. Chem., Vol. 278, Issue 14, 12109-12119, April 4, 2003
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From the The structures of Rhizobium
leguminosarum and Rhizobium etli lipid A are distinct
from those found in other Gram-negative bacteria. Whereas the more
typical Escherichia coli lipid A is a hexa-acylated
disaccharide of glucosamine that is phosphorylated at positions 1 and
4', R. etli and R. leguminosarum lipid A
consists of a mixture of structurally related species (designated A-E) that lack phosphate. A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4' and a
secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2'
acyloxyacyl moiety, is present in all five components. The proximal end
is heterogeneous, differing in the number and lengths of acyl chains
and in the identity of the sugar itself. A proximal glucosamine unit is
present in B and C, but an unusual 2-amino-2-deoxy-gluconate
moiety is found in D-1 and E. We now demonstrate that membranes of
R. leguminosarum and R. etli can convert B to
D-1 in a reaction that requires added detergent and is inhibited by
EDTA. Membranes of Sinorhizobium meliloti and E. coli lack this activity. Mass spectrometry demonstrates that B is
oxidized in vitro to a substance that is 16 atomic mass
units larger, consistent with the formation of D-1. The
oxidation of the lipid A proximal unit is also demonstrated by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry in the positive and negative modes using the model
substrate, 1-dephospho-lipid IVA. With this material, an
additional intermediate (or by product) is detected that is tentatively
identified as a lactone derivative of 1-dephospho-lipid
IVA. The enzyme, presumed to be an oxidase, is located
exclusively in the outer membrane of R. leguminosarum as
judged by sucrose gradient analysis. To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not
been reported previously.
An Outer Membrane Enzyme That Generates the
2-Amino-2- deoxy-gluconate Moiety of Rhizobium
leguminosarum Lipid A*,
,
Department of Biochemistry, Duke University
Medical Center, Durham, North Carolina 27710 and the
§ Department of Pharmacology and Molecular Sciences, The
Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
*
This work was supported by National Institutes of Health
Grants R37-GM-51796 (to C. R. H. R.) and GM-54882 (to
R. J. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental figure.
¶
Present address: Ciphergen Biosystems, Inc., Fremont, CA 94555.
To whom correspondence should be addressed. Tel.:
919-684-5326; Fax: 919-684-8885; E-mail: raetz@biochem.duke.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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