Molecular cloning and characterization of a novel beta-N-acetyl-D-glucosaminidase from Vibrio furnissii.
The accompanying papers (Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33414-33424; Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33425-33432) describe two unique beta-N-acetylglucosaminidases from Vibrio furnissii. A third, ExoII, is reported here. The gene, exoII, was cloned into Escherichia coli, sequenced, and ExoII purified to apparent homogeneity (36 kDa). The molecular weight and N-terminal 16 amino acids of the protein conform to the predicted sequence. ExoII exhibited unique substrate specificity. It rapidly cleaved p-nitrophenyl and 4-methylumbelliferyl beta-GlcNAc, was slightly active with p-nitrophenyl-beta-GalNAc, and was inactive with all other GlcNAc derivatives tested, including N,N'-diacetylchitobiose and (GlcNAc)n, n = 3-6. Unlike GlcNAc (Ki, 210 microM), (GlcNAc)n are poor inhibitors of ExoII. The predicted protein sequence is unique among beta-N-acetylglucosaminidases excepting Cht60, recently cloned from a marine Alteromonas (Tsujibo, H., Fujimoto, K., Tanno, H., Miyamoto, K., Imada, C., Okami, Y., and Inamori, Y. (1994) Gene (Amst.) 146, 111-115). Cht60, a chitobiase, is 26.9% identical to ExoII in a 182-amino acid overlap, but the two enzymes differ in substrate specificity and other properties. ExoII shares similarity with five bacterial and yeast beta-glucosidases, up to 44% identity in the 25-amino acid catalytic domain. By analogy, ExoII may play a role in signal transduction between invertebrate hosts and V. furnissii.
Chitlaru
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E
Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Roseman
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S
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; C014124:4-nitrophenyl-N-acetyl-2-deoxyglucopyranoside; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine