Involvement of an unusual mol operon in molybdopterin cofactor biosynthesis in Ralstonia eutropha

In contrast to its parent strain, transposon Tn5-Mob insertion mutant HB6 of the facultative chemoautotroph Ralstonia eutropha was unable to grow organoautotrophically on formate and exhibited no activity of Mo-dependent, membrane-bound formate dehydrogenase (M-FDH) when cultivated mixotrophically on fructose plus formate. The activity of another molybdoenzyme, the soluble, NAD+-linked formate dehydrogenase which is the key enzyme of formate utilization in R. eutropha, was greatly diminished in the mutant. HB6 also lacked the W-dependent M-FDH activities that were newly discovered in organoautotrophically, lithoautotrophically, or mixotrophically grown wildtype cells. However, an additional W-dependent M-FDH activity, observed in heterotrophically grown stationary-phase cells, was present in the mutant although at a considerably reduced level. Sequence analyses of the complementing chromosomal wildtype and the corresponding mutant DNA fragment revealed the transposon insertion to be located in moeA, a gene involved in the biosynthesis of the molybdopterin cofactor (MoCo). Nevertheless, mutant HB6 was able to grow on xanthine as carbon and energy source and with nitrate as nitrogen source. The utilization of these substrates requires the function of the MoCo-containing enzymes xanthine dehydrogenase and assimilatory nitrate reductase, respectively, that were still active in the mutant. A moeA deletion mutant exhibited the same phenotype as that of HB6. The moeA gene belongs to an unusual mol operon consisting of four genes (moeA, moaD, moaE, and moaF) and being constitutively expressed at low level. Unlike MoeA, the large subunit of molybdopterin synthase encoded by moaE is essential for molybdopterin biosynthesis as was evident by the phenotype of a moaE deletion mutant. MoaF is a novel gene product which showed no similarity to proteins with known function but was indispensable for reconstituting organoautotrophic growth in HB6. The findings suggest that MoeA of R. eutropha is differentially involved in the biosynthesis or incorporation of pterin cofactors of/into the various molybdo- and tungstoenzymes.