Characterization of Escherichia coli MoeB and Its Involvement in the Activation of Molybdopterin Synthase for the Biosynthesis of the Molybdenum Cofactor

Abstract

Amino acid sequence comparisons of Escherichia coliMoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitinactivating step in the ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression. Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration. Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeBbound sulfur during the sulfuration of MoaD. Amino acid substitutions were generated in every cysteine residue in MoeB. All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs. For these cysteines, loss of activity correlated with loss of metal binding.In Escherichia coli, several loci (moa, mob, mod, moe, and mog) have been implicated in the pleiotrophy of the molybdenum enzymes. With the exception of mod, all of these are involved in the biosynthesis of the molybdenum cofactor (1). Molybdenum cofactor contains a tricyclic pterin derivative termed molybdopterin (MPT) 1 that bears the cis-dithiolene group essential for molybdenum ligation. The dithiolene group is generated by MPT synthase, a heterotetrameric protein consisting of two large MoaE subunits (16.9 kDa) and two small MoaD subunits (8.8 kDa) (2). Mass spectrometry has identified a thiocarboxylate in the activated form of MoaD that serves as the sulfur donor for the synthesis of MPT from precursor Z. This reaction can be carried out in vitro with the purified components (3). The high-resolution crystal structure of MPT synthase revealed that the C terminus of each MoaD subunit is inserted into a MoaE subunit to form the active site (2). In addition, the small subunit of MPT synthase shows high structural similarity to the eukaryotic protein ubiquitin (2). The observation that an E. coli moeB mutant accumulates precursor Z indicated the absence of an active form of MPT synthase in the mutant (4). This led to the conclusion that MoeB is the MPT synthase sulfurase, the protein responsible for regenerating the thiocarboxylate group at the C terminus of MoaD in an ATP-dependent reaction (5).Significant sequence similarities between MoeB and a number of other proteins have been identified (6). Particularly noteworthy is the amino acid sequence identity of 23% between MoeB and the eukaryotic ubiquitin-activating enzyme E1, encoded by Uba1 (5). As part of the process for ubiquitin-targeted degradation of proteins, Uba1 was shown to activate ubiquitin in an ATP-dependent reaction with the initial formation of an Uba1-ubiquitin ade...

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans

“…Functional complementation studies of an E. coli moeB mutant strain with MOCS2A, MOCS2B, and MOCS3-C239A showed that the level of nitrate reductase activity was 81% reduced compared to wild-type MOCS3. In contrast, substitution of the corresponding amino acid residue C187 in E. coli MoeB resulted only in a slightly reduced activity of the protein, consistent with the results obtained from in vitro studies (10). This shows that C239 is more important for the sulfur transfer reaction in human MOCS3 than the corresponding residue in E. coli MoeB.…”

“…We have observed that inorganic sulfide can substitute for sulfurated MOCS3-RLD in the thiocarboxylation of MOCS2A in vitro (data not shown) as observed earlier in an in vitro system using the E. coli proteins (10).…”

“…So far, nothing is known about the sulfur donor or the mechanism of sulfur transfer to MPT synthase in humans. In contrast, the reaction mechanism of resulfuration of E. coli MPT synthase has been described in detail (8)(9)(10)(11). Similar to ubiquitinactivating enzymes (E1), E. coli MoeB, the MPT synthase sulfurase, activates the C terminus of MoaD to form an acyl adenylate.…”

Mechanistic Studies of Human Molybdopterin Synthase Reaction and Characterization of Mutants Identified in Group B Patients of Molybdenum Cofactor Deficiency

“…MoaD, the small subunit of E. coli MPT synthase, shows a three-dimensional fold similar to ubiquitin and interacts via its C terminus with the large subunit, MoaE, thereby forming two hypothetical active sites in the heterotetramer (14,15). Prior to the formation of the MoaD C-terminal thiocarboxylate group, the protein is activated by adenylation of the C-terminal carboxylate, a reaction carried out by the E. coli MoeB protein (16,17). Subsequently, the MoaD thiocarboxylate group is formed by the action of a NifS-like protein using L-cysteine as the ultimate sulfur source (18).…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…Although the highly-labile and complex MobisPGD cofactor is an essential component for DMSO reductase activity, understanding its insertion in the maturation pathway poses as a challenge to understanding CISM folding. Currently, it is postulated that MoeB and MoaA are involved in the final cofactor hand-off during CISM maturation (60)(61)(62)(63).…”