Characterization of a novel D-lyxose isomerase from Cohnella laevoribosii RI-39 sp. nov.
A newly isolated bacterium, Cohnella laevoribosii RI-39, could grow in a defined medium with L-ribose as the sole carbon source. A 21-kDa protein isomerizing L-ribose to L-ribulose, as well as D-lyxose to D-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (lyxA) encoding D-lyxose (L-ribose) isomerase was cloned and expressed in Escherichia coli. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn(2+). C. laevoribosii D-lyxose (L-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70 degrees C in the presence of Mn(2+) for D-lyxose and L-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for D-lyxose, L-ribose, and D-mannose, with apparent K(m) values of 22.4 +/- 1.5 mM, 121.7 +/- 10.8 mM, and 34.0 +/- 1.1 mM, respectively. The catalytic efficiencies (k(cat)/K(m)) of CLLI were 84.9 +/- 5.8 mM(-1) s(-1) for D-lyxose (V(max), 5,434.8 U mg(-1)), 0.2 mM(-1) s(-1) for L-ribose (V(max), 75.5 +/- 6.0 U mg(-1)), and 1.4 +/- 0.1 mM(-1) s(-1) for D-mannose (V(max), 131.8 +/- 7.4 U mg(-1)). The ability of lyxA to permit E. coli cells to grow on D-lyxose and L-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.
Cho
Eun-Ah
EA
Department of Biotechnology, Yonsei University, Seoul 120-749, Korea.
Lee
Dong-Woo
DW
Cha
Yun-Hwan
YH
Lee
Sang-Jae
SJ
Jung
Heung-Chae
HC
Pan
Jae-Gu
JG
Pyun
Yu-Ryang
YR
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D045482:Hot Springs / Q000382:microbiology; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism*
D014994:Xylose; D007535:Isomerases