Structure and mechanism of action of a cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus with broad specificity phosphatase activity.
The crystal structure of Bacillus stearothermophilus PhoE (originally termed YhfR), a broad specificity monomeric phosphatase with a molecular mass of approximately 24 kDa, has been solved at 2.3 A resolution in order to investigate its structure and function. PhoE, already identified as a homolog of a cofactor-dependent phosphoglycerate mutase, shares with the latter an alpha/beta/alpha sandwich structure spanning, as a structural excursion, a smaller subdomain composed of two alpha-helices and one short beta-strand. The active site contains residues from both the alpha/beta/alpha sandwich and the sub-domain. With the exception of the hydrophilic catalytic machinery conserved throughout the cofactor-dependent phosphoglycerate mutase family, the active-site cleft is strikingly hydrophobic. Docking studies with two diverse, favored substrates show that 3-phosphoglycerate may bind to the catalytic core, while alpha-napthylphosphate binding also involves the hydrophobic portion of the active-site cleft. Combining a highly favorable phospho group binding site common to these substrate binding modes and data from related enzymes, a catalytic mechanism can be proposed that involves formation of a phosphohistidine intermediate on His10 and likely acid-base behavior of Glu83. Other structural factors contributing to the broad substrate specificity of PhoE can be identified. The dynamic independence of the subdomain may enable the active-site cleft to accommodate substrates of different sizes, although similar motions are present in simulations of cofactor-dependent phosphoglycerate mutases, perhaps favoring a more general functional role. A significant number of entries in protein sequence databases, particularly from unfinished microbial genomes, are more similar to PhoE than to cofactor-dependent phosphoglycerate mutases or to fructose-2,6-bisphosphatases. This PhoE structure will therefore serve as a valuable basis for inference of structural and functional characteristics of these proteins.
Rigden
Daniel J
DJ
National Centre of Genetic Resources and Biotechnology, Cenargen/Embrapa, S.A.I.N. Parque Rural, Final W5, Asa Norte, Brasília, 70770-900, Brazil.
Mello
Luciane V
LV
Setlow
Peter
P
Jedrzejas
Mark J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism*; D018360:Crystallography, X-Ray; D030562:Databases, Protein; D019143:Evolution, Molecular; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006801:Humans; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010736:Phosphoglycerate Mutase / Q000737:chemistry* / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D012984:Software; D013379:Substrate Specificity
D003067:Coenzymes; D008024:Ligands; D010744:Phosphoric Monoester Hydrolases; D010736:Phosphoglycerate Mutase