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Originally published In Press as doi:10.1074/jbc.M001042200 on July 25, 2000

J. Biol. Chem., Vol. 275, Issue 42, 33077-33083, October 20, 2000
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Chitin Catabolism in the Marine Bacterium Vibrio furnissii
IDENTIFICATION, MOLECULAR CLONING, AND CHARACTERIZATION OF A N,N'-DIACETYLCHITOBIOSE PHOSPHORYLASE*

Jae Kweon Park, Nemat O. KeyhaniDagger , and Saul Roseman§

From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

The major product of bacterial chitinases is N,N'-diacetylchitobiose or (GlcNAc)2. We have previously demonstrated that (GlcNAc)2 is taken up unchanged by a specific permease in Vibrio furnissii (unlike Escherichia coli). It is generally held that marine Vibrios further metabolize cytoplasmic (GlcNAc)2 by hydrolyzing it to two GlcNAcs (i.e. a "chitobiase "). Here we report instead that V. furnissii expresses a novel phosphorylase. The gene, chbP, was cloned into E. coli; the enzyme, ChbP, was purified to apparent homogeneity, and characterized kinetically. The DNA sequence indicates that chbP encodes an 89-kDa protein. The enzymatic reaction was characterized as follows.
(<B><UP>GlcNAc</UP></B>)<SUB><B><UP>2</UP></B></SUB>+<B><UP>P</UP></B><SUB><B><UP>i</UP></B></SUB> ⇌ <B><UP>GlcNAc</UP></B>-<B><UP>&agr;-1-P</UP></B>+<B><UP>GlcNAc</UP></B>

K′<SUB><B><UP>cq</UP></B></SUB>=<B><UP>1.0±0.2</UP></B>

<SC>Reaction</SC> 1
The Km values for the four substrates were in the range 0.3-1 mM. p-Nitrophenyl-(GlcNAc)2 was cleaved at 8.5% the rate of (GlcNAc)2, and p-nitrophenyl (PNP)-GlcNAc was 36% as active as GlcNAc in the reverse direction. All other compounds tested displayed <= 1% of the activity of the indicated substrates including: for phosphorolysis, higher chitin oliogsaccharides, (GlcNAc)n, n = 3-5, cellobiose, PNP-GlcNAc, and PNP-(GlcNAc)3; for synthesis, (GlcNAc)n (n = 2-5), glucose, etc. (GlcNAc)2 is a major regulator of the chitin catabolic cascade. Conceivably GlcNAc-alpha -1-P plays a similar but different role in regulation.

* This work was supported by Grant GM51215 from the National Institutes of Health.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF230379.

Dagger Present address: Dept. of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611.

§ To whom correspondence should be addressed: Dept. of Biology and the McCollum-Pratt Inst., Johns Hopkins University, Mudd Hall, Rm. 214, 3400 N. Charles St., Baltimore, MD 21218.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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