Chitin catabolism in the marine bacterium Vibrio furnissii. Identification, molecular cloning, and characterization of A N, N'-diacetylchitobiose phosphorylase.
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. (GlcNAc)(2)+P(i) GlcNAc-alpha-1-P+GlcNAc K'(cq)=1.0+/-0.2 Reaction 1 The K(m) 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.
Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
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Park
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Keyhani
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Roseman
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002686:Chitin / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003360:Cosmids; D004187:Disaccharides / Q000378:metabolism*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004187:Disaccharides; D011994:Recombinant Proteins; D002686:Chitin; C010913:N,N-diacetylchitobiose; C415834:N,N'-diacetylchitobiose phosphorylase; D017351:N-Acetylglucosaminyltransferases; D000117:Acetylglucosamine