ABSTRACT
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 Mn2+. C. laevoribosii
d-lyxose (l-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70°C in the presence of Mn2+ 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 Km
values of 22.4 ± 1.5 mM, 121.7 ± 10.8 mM, and 34.0 ± 1.1 mM, respectively. The catalytic efficiencies (k
cat/Km
) 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.
Characterization of the all‐
E. coli
transcription‐translation system myTXTL by mass spectrometry