title: Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.
abstract: We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces lividans. This was accomplished by using the vector pIJ702 and BglII-BclI libraries of M. viridifaciens chromosomal inserts created in S. lividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BglII fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M(r) of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. lividans.
authors.affiliation: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Japan.
authors.forename: K
authors.initials: K
authors.lastname: Sakurada
authors.forename: T
authors.initials: T
authors.lastname: Ohta
authors.forename: M
authors.initials: M
authors.lastname: Hasegawa
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D008847:Micromonospora / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics* / Q000302:isolation & purification; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012097:Repressor Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics / Q000254:growth & development; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase