Molecular Cloning and Characterization of a Novel β-N-Acetyl-D-glucosaminidase from Vibrio furnissii

Abstract

) describe two unique ␤-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 ␤-GlcNAc, was slightly active with p-nitrophenyl-␤-GalNAc, and was inactive with all other GlcNAc derivatives tested, including N,N-diacetylchitobiose and (GlcNAc) n , n ‫؍‬ 3-6. Unlike GlcNAc (K i , 210 M), (GlcNAc) n are poor inhibitors of ExoII.The predicted protein sequence is unique among ␤-Nacetylglucosaminidases 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 ␤-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.The chitin catabolic cascade of the marine bacterium Vibrio furnissii comprises a complex array of genes and gene products required for the utilization of chitin. The accompanying papers describe the molecular cloning and characterization of two hexosaminidases that are part of this cascade (1, 2).In this report we describe the molecular cloning of a thirdThis novel enzyme differs from all reported ␤-GlcNAcidases, possibly excepting one, in its substrate specificity and amino acid sequence. The enzyme is designated ExoII to differentiate it from the periplasmic exohexosaminidase, ExoI (2); the corresponding structural genes are exoI and exoII, respectively. EXPERIMENTAL PROCEDURES MaterialsThe following chemicals were purchased or were gifts from the indicated sources. Chitin, GlcNAc, 2 p-nitrophenyl (PNP) glycosides, and GlcNAc-6-P were from Sigma; chitin oligosaccharides, (GlcNAc) n (n ϭ 2-6), were from Seikagaku America, Inc. (Rockville, MD); MUF-␤-GlcNAc and MUF-␤-(GlcNAc) 2 were from Calbiochem; phenyl-␤-GlcNAc, benzyl-␤-GlcNAc, p-methylphenyl-␤-GlcNAc, 6-aminohexyl-␤-GlcNAc, ␤-GlcNAc-Asn, and Thr/Ser-Asn-GlcNAc-GlcNAc-(Man) 9 were kindly provided by Dr. Y. C. Lee (Department of Biology, The Johns Hopkins University, Baltimore, MD). . Reagents for molecular biology were obtained from New England Biolabs, U. S. Biochemical Corp., Life Technologies, Inc., Stratagene, and Boehringer Mannheim. Vector pVEX-11 was a kind gift from Dr. Chaudhary (Laboratory of Molecular Biology, National Institutes of Health, Bethesda, MD). Primers T7, T3 were purchased from Stratagene. Additional primers were synthesized in the laboratory of Dr. Robert Schleif (Department of Biology, The Johns Hopkins University, Baltimore, MD). Radioisotopes...

Molecular Characterization of an Intracellular β-N-Acetylglucosaminidase Involved in the Chitin Degradation System ofStreptomyces thermoviolaceusOPC-520

“…On the other hand, Chitlaru and Roseman also reported that -N-acetylglucosaminidase (ExoII) from V. furnissii is a cytoplasmic enzyme which has no activity towards any of the chitin oligosaccharides. 33) They speculated that ExoII might produce phenol derivatives that induce V. furnissii to invade invertebrate cuticles. Therefore, NagC is the first reported example of a cytoplasmic -N-acetylglucosaminidase involved in the chitin degradation system.…”

Identification of a Catalytic Residue ofClostridium paraputrificumN-Acetyl-β-D-glucosaminidase Nag3A by Site-Directed Mutagenesis

“…In this enzyme, Asp285 in the N-terminal domain and Glu491 in the C-terminal domain are considered to be involved in catalysis as nucleophile and proton donor, respectively. [12][13][14] Based on this finding, a putative active site of Nags in this family was also deduced to be Asp242 of Vibrio furnissii ExoII 10) and Asp303 of C. paraputrificum Nag3A, 6) corresponding to Asp285 of ExoI. 12) However, another active site, a putative proton donor, was not expected in single-domain enzymes, including C. paraputrificum Nag3A, from the information about barley ExoI, since Nag3A and other single-domain enzymes of family 3 did not contain a second domain.…”

“…6,[9][10][11] From the alignment of the catalytic domains of the 12 Nags (Fig. 1), it can be seen that two Glu residues and six Asp residues are highly conserved among them and are candidates for catalytic amino acid residues.…”

Production of N-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

“…The enzymatic breakdown of chitin by chitinases has been reported from our laboratory [9,10] as well as from others [11][12][13][14][15]. The chitinase gene (chi) of Enterobacter sp.…”