The activation of ubiquitin and related protein modifiers 1 is catalyzed by members of the E1 enzyme family, which utilize ATP for the covalent self-attachment of the modifiers to a conserved cysteine. The Escherichia coli MoeB and MoaD proteins are involved in molybdenum cofactor (Moco) biosynthesis, an evolutionarily conserved pathway 2 . The MoeB-and E1-catalyzed reactions are mechanistically similar, and despite a lack of sequence similarity, MoaD and ubiquitin display the same fold including a conserved C-terminal Gly-Gly motif 3 . Similar to the E1 enzymes, MoeB activates the C-terminus of MoaD to form an acyl-adenylate. Subsequently, a sulfurtransferase converts the MoaD acyladenylate to a thiocarboxylate that acts as the sulfur donor during Moco biosynthesis 4 . These findings suggest that ubiquitin and E1 are derived from two ancestral genes closely related to moaD and moeB 2 . The crystal structures of the MoeB-MoaD complex in its apo, ATP-bound, and MoaD-adenylate forms presented here highlight the functional similarities between the MoeBand E1-substrate complexes. These structures provide a molecular framework for understanding the activation of ubiquitin, Rub, SUMO, and the sulfur incorporation step during Moco and thiamine biosynthesis.The crystal structure of MoeB-MoaD was solved by multiple isomorphous replacement (MIR) using xray diffraction data collected at beamline X26C at the National Synchrotron Light Source at Brookhaven National Laboratory. The complex between the Escherichia coli MoeB and MoaD proteins reveals a MoeB 2 -MoaD 2 heterotetramer (Fig. 1a) in which the MoeB subunits form a dimer. This dimer interface is primarily hydrophobic and buries a surface area of 5,400 Å 2 . To distinguish between the different subunits in the complex, residue numbers are prefixed with either B or D to indicate their location in MoeB or MoaD, respectively.

Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center.

The early steps in the biosynthesis of the molybdopterin portion of the molybdenum cofactor have been investigated through the use of radiolabeled precursors. Labeled guanosine was added to growing cultures of the molybdopterin-deficient Escherichia coli mutant, moeB, which accumulates large amounts of precursor Z, the final intermediate in molybdopterin biosynthesis (Wuebbens, M. M., and Rajagopalan, K. V. (1993) J. Biol. Chem. 268, 13493-13498). Precursor Z is readily oxidized to the stable, fluorescent pterin, compound Z, which contains all 10 of the carbon atoms present in molybdopterin. For these experiments, compound Z was isolated from both the cells and culture media and analyzed for the presence of label. The development of a method for sequential cleavage of the compound Z side chain carbons facilitated determination of the distribution of label between the ring and the side chain of compound Z. Addition of uniformly labeled [14C]guanosine to moeB cultures produced compound Z labeled in both the ring and the side chain. Growth on [8-14C]guanosine resulted in transfer of label to the C-1' position of compound Z. The label present in compound Z purified from cultures grown on [8,5'-3H]guanosine was lost by removal of the three terminal side chain carbons. These results indicate that although a guanosine compound serves as the initial precursor for molybdopterin biosynthesis, the early steps of this pathway in E. coli proceed via a pathway unlike that of any known pteridine biosynthetic pathway.