Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, from Klebsiella sp. 601.
A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat//Km values of 0.49 mmol/L, 1.08 x 103 s-1, and 2.23 x 103 s-1 x mmol/L-1, respectively, for DMP, 5.63 mmol/L, 6.64 x 103 s-1, and 1.18 x 103 s-1 x mmol/L-1 for ABTS, and 0.023 mmol/L, 11 s-1, and 4.68 x 102 s-1 x mmol/L-1 for SGZ.
Li
Yang
Y
Faculty of Life Sciences, Hubei University, Wuhan 430062, China.
Yin
Jiao
J
Qu
Guosheng
G
Lv
Luchao
L
Li
Yadong
Y
Yang
Sheng
S
Wang
Xing-Guo
XG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D007709:Klebsiella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012988:Soil Microbiology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003300:Copper; D010088:Oxidoreductases