Molecular analysis of the gene encoding a novel transglycosylative enzyme from Alteromonas sp. strain O-7 and its physiological role in the chitinolytic system.
We purified from the culture supernatant of Alteromonas sp. strain O-7 and characterized a transglycosylating enzyme which synthesized beta-(1-->6)-(GlcNAc)2, 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2- deoxyglucopyranose from beta-(1-->4)-(GlcNAc)2. The gene encoding a novel transglycosylating enzyme was cloned into Escherichia coli, and its nucleotide sequence was determined. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 99,560 Da which corresponds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the cloned enzyme was much larger than that of enzyme (70 kDa) purified from the supernatant of this strain. These results suggest that the native enzyme was the result of partial proteolysis occurring in the N-terminal region. The enzyme showed significant sequence homology with several bacterial beta-N-acetylhexosaminidases which belong to family 20 glycosyl hydrolases. However, this novel enzyme differs from all reported beta-N-acetylhexosaminidases in its substrate specificity. To clarify the role of the enzyme in the chitinolytic system of the strain, the effect of beta-(1-->6)-(GlcNAc)2 on the induction of chitinase was investigated. beta-(1-->6)-(GlcNAc)2 induced a level of production of chitinase similar to that induced by the medium containing chitin. On the other hand, GlcNAc, (GlcNAc)2, and (GlcNAc)3 conversely repressed the production of chitinase to below the basal level of chitinase activity produced constitutively in medium without a carbon source.
Tsujibo
H
H
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. tsujibo@oysun01.oups.ac.jp
Kondo
N
N
Tanaka
K
K
Miyamoto
K
K
Baba
N
N
Inamori
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000096:biosynthesis; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004790:Enzyme Induction; D005798:Genes, Bacterial; D006031:Glycosylation; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D004269:DNA, Bacterial; D002686:Chitin; D002688:Chitinases; D001619:beta-N-Acetylhexosaminidases