A novel beta-N-acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: gene cloning, expression, and assignment to family 3 of the glycosyl hydrolases.
A beta-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66, 329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned beta-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical beta-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable beta-glucosidase activity, revealed homology with microbial beta-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of beta-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60 degreesC and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-beta-N-acetylglucosamine were 425.7 microM and 24.8 micromol min-1 mg of protein-1, respectively.
Tsujibo
H
H
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. tsujibo@oysun01.oups.ac.jp
Hatano
N
N
Mikami
T
T
Hirasawa
A
A
Miyamoto
K
K
Inamori
Y
Y
D000118:Acetylglucosaminidase / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D000118:Acetylglucosaminidase