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 F 420 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 micromol min (-1) mg (-1), GTP K M (app) = 56 microM, and k cat/ K M (app) = 2 x 10 (4) M (-1) s (-1) and LP K M (app) = 36 microM, 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.
Grochowski
Laura L
LL
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0308, USA.
Xu
Huimin
H
White
Robert H
RH
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000096:biosynthesis / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007773:Lactates / Q000737:chemistry / Q000378:metabolism*; D017015:Methanococcales / Q000201:enzymology / Q000378:metabolism; D015394:Molecular Structure; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity
D019843:Archaeal Proteins; D003067:Coenzymes; D007773:Lactates; C048984:2-phospholactic acid; C007701:factor 420; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D012256:Riboflavin