Crystal structure of the molybdenum cofactor biosynthesis protein MobA from Escherichia coli at near-atomic resolution

Background: All mononuclear molybdoenzymes bind molybdenum in a complex wit h an organic cofactor termed molybdopterin (MPT). In many bacteria, includi ng Escherichia coli, molybdopterin can be further modified by attachment of a GMP group to the terminal phosphate of molybdopterin to form molybdopter in guanine dinucleotide (MGD). This modification reaction is required for t he functioning of many bacterial molybdoenzymes, including the nitrate redu ctases, dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) reductas es, and formate dehydrogenases. The GMP attachment step is catalyzed by the cellular enzyme MobA. Results: The crystal structure of the 21.6 kDa E. coli MobA has been determ ined by MAD phasing with selenomethionine-substituted protein and subsequen tly refined at 1.35 Angstrom resolution against native data. The structure consists of a central, predominantly parallel P sheet sandwiched between tw o layers of a helices and resembles the dinucleotide binding Rossmann fold. One face of the molecule bears a wide depression that is lined by a number of strictly conserved residues, and this feature suggests that this is whe re substrate binding and catalysis take place. Conclusions: Through comparisons with a number of structural homologs, we h ave assigned plausible functions to several of the residues that line the s ubstrate binding pocket. The enzymatic mechanism probably proceeds via a nu cleophilic attack by MPT on the GMP donor, most likely GTP, to produce MGD and pyrophosphate. By analogy with related enzymes, this process is likely to require magnesium ions.