Characterization of a Novel d -Lyxose Isomerase from Cohnella laevoribosii RI-39 sp. nov

Abstract

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 Mn 2+ . C. laevoribosii d -lyxose ( l -ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70°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.