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J. Biol. Chem., Vol. 279, Issue 2, 1553-1561, January 9, 2004

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Genes malh and pagl of Clostridium acetobutylicum ATCC 824 Encode NAD⁺- and Mn²⁺-dependent Phospho--glucosidase(s)^*

John Thompson, Sonja Hess¶, and Andreas Pikis||

From the Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, NIDCR and the ^¶Proteomics and Mass Spectrometry Facility, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

The genome of Clostridium acetobutylicum 824 contains two genes encoding NAD⁺, Mn²⁺, and dithiothreitol-dependent phospho--glucosidases that can be assigned to family 4 of the glycosylhydrolase superfamily. The two genes, designated malh (maltose 6-phosphate hydrolase) and pagl (phospho--glucosidase), respectively, reside in separate operons that also encode proteins of the phosphoenolpyruvate-dependent:sugar phosphotransferase system. C. acetobutylicum grows on a variety of -linked glucosides, including maltose, methyl--D-glucoside, and the five isomers of sucrose. In the presence of the requisite cofactors, extracts of these cells readily hydrolyzed the chromogenic substrate p-nitrophenyl--D-glucopyranoside 6-phosphate, but whether hydrolysis reflected expression of enzymes encoded by the malh or pagl genes was not discernible by spectrophotometric analysis or polyacrylamide gel electrophoresis. Resolution of this question required the cloning of the malh and pagl genes, and subsequent high expression, purification, and characterization of maltose-6'-phosphate hydrolase (MalH) and phospho--glucosidase (PagL), respectively. MalH and PagL exhibit 50% residue identity, and in solution are tetramers comprising similar sized (50 kDa) subunits. The two proteins cross-react with polyclonal rabbit antibody against phospho--glucosidase from Fusobacterium mortiferum. Purified MalH and PagL cleaved p-nitrophenyl--D-glucopyranoside 6-phosphate with comparable efficiency, but only MalH catalyzed the hydrolysis of disaccharide 6'-phosphates formed via the phosphoenolpyruvate-dependent:sugar phosphotransferase system. Importantly, analysis of the proteome of C. acetobutylicum 824 by electrospray ionization-mass spectrometry confirmed expression of MalH during growth on many -glucosides tested. Site-directed changes C169S and D170N yielded full-length, but catalytically inactive MalH. Of the two putative operons, our findings suggest that only proteins encoded by the mal operon participate in the dissimilation of maltose and related O--linked glucosides by C. acetobutylicum 824.

Received for publication, September 29, 2003 , and in revised form, October 21, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, MD 20850.

To whom correspondence should be addressed: NIDCR, National Institutes of Health, Bldg. 30, Rm. 528, Convent Dr. MSC-4350, Bethesda, MD 20892. Tel.: 301-496-4083; Fax: 301-402-1064; E-mail: jthompson{at}dir.nidcr.nih.gov.

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