Physiological and Genetic Analyses Leading to Identification of a Biochemical Role for the moeA (Molybdate Metabolism) Gene Product in Escherichia coli

A unique class of chlorate-resistant mutants of Escherichia coli which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the moeA gene. Mutant strains with polar mutations in moeA which are also moeB did not produce active molybdoenzymes in any of the media tested. moeA mutants with a second mutation in either cysDNCJI or cysH gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of moeA cys double mutants suppressed the MoeA⁻ phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.Molybdoenzymes play essential metabolic roles in most organisms from bacteria to plants and animals (34). All molybdoenzymes other than dinitrogenase contain molybdenum cofactor, which consists of a unique molybdopterin (MPT) complexed with molybdenum (1, 12, 23, 31, 34). In Escherichia coli, the biologically active form of the cofactor in molybdoenzymes is MPT guanine dinucleotide (MGD) (5, 22, 23). Synthesis of this cofactor in an active form requires transport of molybdate into the cell, activation of molybdate, synthesis of the MPT moiety, and incorporation of molybdate into MPT. Although molybdate transport and the various steps in the organic part of MGD biosynthesis are well characterized (17, 24, 33; see references 10, 22, and 23 for reviews), very little is known about the activation and incorporation of molybdenum into the cofactor (22).Mutants which are defective in molybdate metabolism can be isolated as chlorate-resistant mutants (8, 9). A large fraction of these mutants are pleiotropic for all molybdoenzyme activities in the cell, and these comprise the three genetic loci involved in MGD synthesis, moa, mob, and moeB (see references 10, 22, 29, and 31 for reviews). The mod gene products comprise the molybdate transport system through which molybdate is transported into the cell and the Mod⁻ phenotype can be suppressed by increasing molybdate concentration in the medium. The mog mutants which produced formate hydrogenlyase (FHL) activity containing the molybdoenzyme formate dehydrogenase-H (FDH-H) but not nitrate reductase activity was proposed to be defective in molybdochelatase (13, 32). This molybdochelatase is apparently required for production of active nitrate reductase and not for FDH-H.The moe operon codes for two proteins, and only the physiological role of the second gene product, MoeB protein, is known. The MoeB protein activates MPT synthase, which catalyzes the conversion of MPT precursor (precursor Z) to MPT by introducing the needed sulfur to which Mo is coordinated in the molybdenum cofactor (20, 22). The MoeB protein, MPT synthase sulfurylase, is the known S donor in the activation of MPT synthase. The physiological role of MoeA protein coded by the first gene in the two member moe operon is not known. Mutants which are defective in moeA (chlE [29]) produced about 6% of the wild-type levels of MPT (12), although no molybdoenzyme activity was found in these moeA mutants. Since the MoeB protein acts as an S donor in MPT synthesis, it is possible that the first gene product, MoeA protein, also has a similar role in linking S metabolism and Mo metabolism in the cell.During our analysis of molybdate transport-defective mutants, we identified a subgroup of chlorate-resistant mutants with a unique phenotype. Mutations in this class of mutants were mapped in the moeA gene at 18.6 min on the E. coli chromosome (3, 18). The MoeA⁻ phenotype was suppressed when the growth medium was supplemented with sulfide. In this report, we present the physiological and genetic characteristics of E. coli moeA mutants and propose a role for the MoeA protein in the activation of molybdenum by sulfurylation.(This work was presented at the International Symposium on Nitrogen Assimilation: Molecular and Genetic Aspects, 3 to 9 May 1997, Tampa, Fla.)