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J. Biol. Chem., Vol. 277, Issue 36, 32606-32615, September 6, 2002
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From the Bacterial sulfate assimilation pathways provide
for activation of inorganic sulfur for the biosynthesis of cysteine and
methionine, through either adenosine 5'-phosphosulfate (APS) or
3'-phosphoadenosine 5'-phosphosulfate (PAPS) as intermediates. PAPS is
also the substrate for sulfotransferases that produce sulfolipids,
putative virulence factors, in Mycobacterium tuberculosis
such as SL-1. In this report, genetic complementation using
Escherichia coli mutant strains deficient in APS kinase and
PAPS reductase was used to define the M. tuberculosis
and Mycobacterium smegmatis CysH enzymes as APS reductases.
Consequently, the sulfate assimilation pathway of M. tuberculosis proceeds from sulfate through APS, which is acted on
by APS reductase in the first committed step toward cysteine and
methionine. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. Deletion of
CysH from M. smegmatis afforded a cysteine and methionine
auxotroph consistent with a metabolic branch point centered on APS. In
addition, we have redefined the substrate specificity of the B. subtilis CysH, formerly designated a PAPS reductase, as an APS
reductase, based on its ability to complement a mutant E. coli strain deficient in APS kinase. Together, these studies show
that two conserved sequence motifs, CCXXRKXXPL
and SXGCXXCT, found in the C termini of all APS
reductases, but not in PAPS reductases, may be used to predict the
substrate specificity of these enzymes. A functional domain of the
M. tuberculosis CysC protein was cloned and expressed in
E. coli, confirming the ability of this organism to make
PAPS. The expression of recombinant M. tuberculosis APS
kinase provides a means for the discovery of inhibitors of this enzyme
and thus of the biosynthesis of SL-1.
5'-Adenosinephosphosulfate Lies at a Metabolic Branch Point
in Mycobacteria*
§,
,**
Howard Hughes Medical Institute and
Departments of Chemistry and Molecular and Cell Biology, University
of California, Berkeley, California 94720 and the ¶ School of
Public Health, University of California,
Berkeley, California 94720
*
This work was supported by National Institutes of Health
(NIH) Grants GM59907 and AI51622. Sequencing of M. avium and
M. smegmatis was accomplished with support from NIAID, NIH.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.
Recipient of a Ford Foundation graduate Fellowship.
**
To whom correspondence should be addressed. E-mail:
bertozzi@cchem.berkeley.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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