Biochemical and Structural Analysis of the Molybdenum Cofactor Biosynthesis Protein MobA

Annika Guse,Clare E.M Stevenson,Jochen Kuper,Grant Buchanan,Günter Schwarz,Gérard Giordano,Axel Magalon,Ralf R Mendel,David M Lawson,Tracy Palmer

doi:10.1074/jbc.m302639200

Abstract

Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.

Highlights

The biosynthesis of the molybdenum cofactor (Moco) can be divided into three stages
Molybdopterin guanine dinucleotide (MGD) is the most of the proteins involved in MPT synthesis have been form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes
MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals used were from the highest grade available. Xanthine oxidase (EC 1.1.3.22) from buttermilk grade I was obtained from Sigma. The crude cell extract, which was prepared after centrifugation [26], was loaded onto a 1 ml of nickelnitrilotriacetic acid column that had been previously pre-equilibrated with washing buffer (50 mM sodium phosphate, pH 6.0, 300 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol). MobA proteins were overproduced in strain M15[pREP4] and purified as described previously [20]. Total molybdenum cofactor (MPT plus MGD) was determined by boiling purified MobA fractions in the presence of acidic iodine. Separate MPT and MGD content of purified protein was determined after room temperature oxidation with acidic iodine. Both methods were as described previously [31].

RESULTS

PDB accession code

Amount of bound MGD

DISCUSSION