Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23.
The nitrile hydratase (NHase) of Pseudomonas chlororaphis B23, which is composed of two subunits, alpha and beta, catalyzes the hydration of nitrile compounds to the corresponding amides. The NHase gene of strain B23 was cloned into Escherichia coli by the DNA-probing method with the NHase gene of Rhodococcus sp. strain N-774 as the hybridization probe. Nucleotide sequencing revealed that an amidase showing significant similarity to the amidase of Rhodococcus sp. strain N-774 was also coded by the region just upstream of the subunit alpha-coding sequence. In addition to these three proteins, two open reading frames, P47K and OrfE, were found just downstream of the coding region of subunit beta. The direction and close locations to each other of these open reading frames encoding five proteins (amidase, subunits alpha and beta, P47K, and OrfE, in that order) suggested that these genes were cotranscribed by a single mRNA. Plasmid pPCN4, in which a 6.2-kb sequence covering the region coding for these proteins is placed under control of the lac promoter, directed overproduction of enzymatically active NHase and amidase in response to addition of isopropyl-beta-D-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of subunits alpha and beta of NHase was about 10% of the total cellular proteins and that an additional 38-kDa protein probably encoded by the region upstream of the amidase gene was also produced in a large amount. The 38-kDa protein, as well as P47K and OrfE, appeared to be important for efficient expression of NHase activity in E. coli cells, because plasmids containing the NHase and amidase genes but lacking the region coding for the 38-kDa protein or the region coding for P47K and OrfE failed to express efficient NHase activity.
Nishiyama
M
M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Horinouchi
S
S
Kobayashi
M
M
Nagasawa
T
T
Yamada
H
H
Beppu
T
T
D000581:Amidohydrolases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics*; D015183:Restriction Mapping; D012240:Rhodococcus / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D015342:DNA Probes; D000581:Amidohydrolases; C019665:amidase; D006836:Hydro-Lyases; C048831:nitrile hydratase