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<p>The molybdenum cofactor (Moco) is an essential enzyme cofactor found in all kingdoms of life. Moco plays central roles in many vital biological processes, and must be biosynthesized de novo. During its biosynthesis, the characteristic pyranopterin ring of Moco is constructed by a complex rearrangement of guanosine 5'-­triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC. However, the mechanisms and the functions of the two enzymes are under significant debate. To elucidate their physiological roles, I took a multidisciplinary approach to functionally characterize MoaA and MoaC in vivo and in vitro. In this dissertation, I report the first isolation and characterization of the physiological MoaC substrate, 3',8-­ cyclo-­7,8-­dihydro-­guanosine 5'-triphosphate (3',8-cH2GTP). I also report the first X-­ray crystal structures of MoaC in complex with this highly air sensitive substrate, and its product cPMP. These studies, combined with in vitro experiments using substrate analogs, catalytically impaired mutants, and synthetic peptides, have enabled me to delineate the functions of the Moco biosynthetic enzymes, MoaA and MoaC, and proposed mechanistic models for their roles in the formation of cPMP.</p> / Dissertation

We have carried out structural studies on bovine pancreatic phospholipase A2 (BPLA2) and two proteins involved in molybdenum cofactor (Moco) biosynthesis pathway. In addition, molecular-dynamics simulations and other analyses have been performed to corroborate the findings obtained from the crystal structures. Crystal structures of the three active-site mutants (H48N, D49N and D49K) of BPLA2 were determined to understand the mechanism by which the mutant H48N is able to catalyze the reaction of phospholipid hydrolysis and to see the effect of the loss of Ca 2+ ion in the active site of D49N and D49K mutants. We found that Asp49 could possibly play the role of a general base instead of His48 in the case of the H48N mutant. In the case of D49N and D49K mutants, the active site of the enzyme is perturbed, whereas the overall tertiary structure of these mutants is intact. In addition, a total of 24 invariant water molecules were identified in all of the crystal structures of BPLA2 available in its archive, PDB. Out of these, four water molecules are essential for the catalytic activity, whereas, the remaining water molecules play a role in the stability of the enzyme. In addition, structural studies on two proteins MoaC and MogA involved in Moco biosynthesis pathway have been carried out. For the first time, crystal structure of MoaC bound with GTP molecule has been reported. The gene id TTHA0341, which is mentioned as MoaB in the CMR database, was annotated as MogA based the comparative analysis of sequences and structures (with the present work and the structures available in the literature). The role of N-and C-termini of MoaB and MogA proteins were proposed that these residues might stabilize the substrate and/or product molecule in the active site. In addition, the residues involved in the oligomerization are compared with MD simulations. The molecular docking studies show that MoaB proteins show more preference to GTP than ATP. The comparison of the two active (MPT and AMP-binding) sites revealed that MPT-binding site is preferred over AMP-binding site for nucleotide binding.