High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system.
N-substituted formamide deformylase (NfdA) from Arthrobacter pascens F164 is a novel deformylase involved in the metabolism of isonitriles. The enzyme catalyzes the deformylation of an N-substituted formamide, which is produced from the corresponding isonitrile, to yield the corresponding amine and formate. The nfdA gene from A. pascens F164 was cloned into different types of expression vectors for Escherichia coli and Streptomyces strains. Expression in E. coli resulted in the accumulation of an insoluble protein. However, Streptomyces strains transformed with a P(nitA)-NitR system, which we very recently developed as a regulatory gene expression system for streptomycetes, allowed the heterologous overproduction of NfdA in an active form. When Streptomyces lividans TK24 transformed with pSH19-nfdA was cultured under the optimum conditions, the NfdA activity of the cell-free extract amounted to 8.5 U/mg, which was 29-fold higher than that of A. pascens F164. The enzyme also comprised approximately 20% of the total extractable cellular protein. The recombinant enzyme was purified to homogeneity and characterized. The expression system established here will allow structural analysis and mutagenesis studies of NfdA.
Fukatsu
Hiroshi
H
Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Herai
Sachio
S
Hashimoto
Yoshiteru
Y
Maseda
Hideaki
H
Higashibata
Hiroki
H
Kobayashi
Michihiko
M
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000588:Amines / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology*; D015854:Up-Regulation
D000588:Amines; D011993:Recombinant Fusion Proteins; D000581:Amidohydrolases; C493455:N-substituted formamide deformylase