Identification and characterization of the 2-phospho-L-lactate guanylyltransferase involved in coenzyme F420 biosynthesis

Coenzyme F-420 is a hydride carrier cofactor functioning in methanogenesis. One step in the biosynthesis of coenzyme F420 involves the coupling of 2-phospho-L-lactate (LP) to 7,8-didemethyl-8-hydroxy-5-deazaflavin, the F-420 chromophore. This condensation requires an initial activation of 2-phospho-L-lactate through a pyrophosphate linkage to GMP. Bioinformatic analysis identified an uncharacterized archaeal protein in the Methanocaldococcus jannaschii genome, MJ0887, which could be involved in this transformation. The predicted MJ0887-derived protein has domain similarity with other known nucleotidyl transferases. The MJ0887 gene was cloned and overexpressed, and the purified protein was found to catalyze the formation of lactyl-2-diphospho-5'-guanosine from LP and GTP. Kinetic constants were determined for the MJ0887-derived protein with both LP and GTP substrates and are as follows: V-max = 3 mu mol min(-1) mg(-1), GTP K-M(app) = 56 mu M, and k(cat)/K-M(app) = 2 x 10(4) M-1 s(-1) and Lp K-M(app) = 36 mu M, and k(cat)/K-M(app) = 4 x 10(4) M-1 s(-1). The MJ0887 gene product has been designated CofC to indicate its involvement in the third step of coenzyme F-420 biosynthesis.