Studies on the chitin catabolic cascade in the marine bacterium Vibrio furnissii

The turnover of chitin is an essential component of the carbon and nitrogen cycles in aquatic ecosystems, and is largely mediated by marine bacteria. Vibrio furnissii hydrolyzes chitin to chitin oligosaccharides, (GlcNAc){dollar}sb{lcub}rm n{rcub}{dollar}, which diffuse through porins into the periplasmic space, and are hydrolyzed there to GlcNAc and (GlcNAc){dollar}sb2{dollar}. The latter are transported via specific permeases and converted to cytoplasmic fructose-6-P, acetate and NH{dollar}sb3{dollar}. The chitin catabolic cascade involves a minimum of 20 proteins and 3 signal transduction systems. The present studies report: (a) characterization of two unique V. furnissii periplasmic enzymes, a chitodextrinase and {dollar}beta{dollar}-hexosaminidase ({dollar}beta{dollar}-GlcNAcidase); (b) an outer membrane protein (Omp-CHO), the putative "chitoporin", induced specifically by (GlcNAc){dollar}sb{lcub}rm n{rcub}{dollar}; and (c) the synthesis of GlcNAc analogues, potentially useful for characterizing transport processes and regulation of the chitinolytic pathway.; The {dollar}beta{dollar}-chitodextrinase was purified to apparent homogeneity from an E. coli recombinant. Two close-running enzymatically active bands were detected on SDS-PAGE (apparent M{dollar}sb{lcub}rm r{rcub}, {lcub}sim{rcub}120{dollar}). The N-terminal sequences of the two proteins were identical. However, they lacked 31 residues of the deduced N-terminus (from the DNA sequence of the cloned gene; 1046 amino acids, 113 kDa). The 31 amino acid polypeptide contains the consensus signal sequence for procaryotic periplasmic proteins. The {dollar}beta{dollar}-chitodextrinase was inactive with chitin, glucosamine oligomers, and cleaved (GlcNAc){dollar}sb{lcub}rm n{rcub}{dollar}, n = 4-6, to (GlcNAc){dollar}sb{lcub}rm n{rcub},{dollar} n = 2-3. The enzyme was substrate inhibited at millimolar (GlcNAc){dollar}sb{lcub}rm n{rcub}{dollar}, n = 4-6.; Homogenous {dollar}beta{dollar}-GlcNAcidase was isolated from an E. coli recombinant, M{dollar}sb{lcub}rm r{rcub}{dollar} = 68. The predicted composition from the DNA sequence of the cloned gene is 611 amino acids, 69.4 kDa. The enzyme was not processed; the predicted and observed N-terminal amino acid sequences were identical. The amino acid sequence is homologous to the {dollar}alpha{dollar}- and {dollar}beta{dollar}-polypeptide chains of human hexosaminidase (defective in Tay-Sachs disease). The {dollar}beta{dollar}-GlcNAcidase was optimally active at pH 7-7.5 with (GlcNAc){dollar}sb{lcub}rm n{rcub}{dollar}, n = 3-6, and PNP-GlcNAc, but at pH 5.8 with (GlcNAc){dollar}sb2{dollar}. Thus, at the pH of sea water, the V{dollar}sb{lcub}rm max{rcub}{dollar} for (GlcNAc){dollar}sb2{dollar} is {dollar}le{dollar}2% of (GlcNAc){dollar}sb{lcub}rm n{rcub}{dollar}, explaining how (GlcNAc){dollar}sb2{dollar}, an important inducer, can penetrate the periplasmic space.; These results provide important information on some steps in the chitin catabolic cascade.