Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA.
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.
Departments of Molecular Microbiology and Biological Chemistry, John Innes Centre, Norwich NR4 7UH, United Kingdom.
Annika
A
Guse
Clare E M
CE
Stevenson
Jochen
J
Kuper
Grant
G
Buchanan
Gunter
G
Schwarz
Gerard
G
Giordano
Axel
A
Magalon
Ralf R
RR
Mendel
David M
DM
Lawson
Tracy
T
Palmer
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D005816:Genetic Complementation Test; D005998:Glycine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013466:Sulfurtransferases / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry; D020798:Two-Hybrid System Techniques
D029968:Escherichia coli Proteins; C490938:MobB protein, E coli; C099206:MobB protein, bacteria; D011994:Recombinant Proteins; D015534:Trans-Activators; C095754:mobA protein, E coli; D001224:Aspartic Acid; D001216:Asparagine; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C111302:MoeA protein, E coli; D013466:Sulfurtransferases; D005998:Glycine