Chitin Catabolism in the Marine Bacterium Vibrio furnissii

Abstract

The major product of bacterial chitinases isN,N′-diacetylchitobiose or (GlcNAc)2. We have previously demonstrated that (GlcNAc)2 is taken up unchanged by a specific permease inVibrio 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 thatV. 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+Pi⇌GlcNAc­α­1­P+GlcNAc K′cq=1.0±0.2 REACTION 1 TheKm 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-α-1-P plays a similar but different role in regulation.