Mutational Analysis of Escherichia coli MoeA:  Two Functional Activities Map to the Active Site Cleft^,

Abstract

SUMMARYThe molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, that of metal ligation to molybdopterin (the organic component of the cofactor) to form active cofactor. In Escherichia coli, the MoeA protein mediates Mo ligation to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The xray crystal structures for both proteins have been previously described as well as two essential MogA residues; Asp49 and Asp82. Here we describe the detailed mutational analysis of the MoeA protein.Variants of conserved residues at the putative active site of MoeA were analyzed for loss of function in two different, previously described assays; one employing moeA − crude extracts, and the other utilizing a defined system. Oddly, no correlation was observed between the observed activity in the two assays. In fact, our results showed a general trend towards an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified components assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. Based upon these results, two different functional areas were assigned to regions at or near the MoeA active site cleft. Keywords molybdopterin; MPT; Moco; molybdenum; MoeA; MogA; gephyrin; Cnx1; cinnamon PDB IDs for the variants: 2NQK (D59N); 2NQM (T100A); 2NQN (T100W); 2NQQ (R137Q); 2NQR (D142N); 2NQS (E188A); 2NQU (E188Q); 2NQV (D228A); 2NRO (K279Q); 2NRP (R350A); and 2NRS (S371W) All molybdenum-containing enzymes with the exception of nitrogenase, utilize a molybdenum cofactor (Moco) 1 consisting of a mononuclear Mo atom coordinated via a cis-dithiolene moiety to the organic molecule molybdopterin (MPT). Moco-containing enzymes function as oxidoreductases in a myriad of reactions in carbon, nitrogen, and sulfur cycles (1). In † This work was supported by National Institutes of Health Grant GM00091 (to KVR) and DK 54835 (to HS). * To whom correspondence should be addressed, Phone: (919) Fax: (919) The X-ray crystal structures of E. coli MoeA has previously been solved, and a putative active site assigned (18,19). To gain a more thorough understanding of the mechanism of MoeAmediated molybdenum ligation, a detailed site-directed mutagenesis study of conserved residues at the putative MoeA active site was undertaken. These variants were analyzed for Moco binding and for loss of function in both the moeA − crude extract assay and the fullydefined system. Results from these experiments were utilized to provide the first picture of the distribution of function across the MoeA 3-D structure. MATERIALS AND METHODS Mutagenesis of MoeAUsing the Transformer Site-Directed Mutagenesi...

Splice-specific Functions of Gephyrin in Molybdenum Cofactor Biosynthesis

“…The missing C6 cassette forms large parts of subdomain 2 (Fig. 8B), which forms the upper half of the GephE active site harboring residues important for Moco biosynthesis as shown for the homologous Cnx1 protein (40) as well as MoeA (41). The fact that Geph2 is expressed at similar levels as other variants (including blob formation) points to an overall maintenance of the structural integrity of GephE.…”

Metal insertion into the molybdenum cofactor: product–substrate channelling demonstrates the functional origin of domain fusion in gephyrin

“…In addition, given the known transfer and binding of MPT-AMP in the E domain, MPT-AMP was modelled into the proposed active site of the E domains ( Figure 7B, yellow subunit), which is surrounded by functionally important residues [44]. Crystal structures of gephyrin E domain [39] and E. coli MoeA [44] disclosed that subdomain 1 is highly mobile and hosts additional active-site residues [44]. In our model, those residues face the G-domain-bound MPT-AMP in a conformation that we call a 'closed' state ( Figure 7C, orange and yellow subunits).…”

Aerobic Degradation of Mercaptosuccinate by the Gram-Negative BacteriumVariovorax paradoxusStrain B4

“…Moco is almost ubiquitous in nature, and its biosynthesis, which comprises several steps, is highly conserved (73). In bacteria, such as E. coli, MoeA is required in the final step of Moco biosynthesis for the mononuclear Mo is attached to MPT, and it is enhanced by MogA (58,72). In this study an moeA-homologous gene was insertionally inactivated in several of the MS-negative mutants of strain B4.…”

“…On the other hand, in most bacterial Modependent enzymes, the fusion of a nucleotide monophosphate to the Moco side chain is required. This reaction is catalyzed by MobA, yielding the molybdopterin guanine dinucleotide (MGD) cofactor, which is the active cofactor of most prokaryotic molybdoenzymes (50,58) and is also found in enzymes belonging to the DMSOR family. In this study, a gene coding for a homologue of the MGD biosynthesis protein (MobA) was identified and located adjacent to moeA in the V. paradoxus strain B4 genome.…”