A new member of the alkaline phosphatase superfamily with a formylglycine nucleophile: structural and kinetic characterisation of a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum.
The alkaline phosphatase superfamily comprises a large number of hydrolytic metalloenzymes such as phosphatases and sulfatases. We have characterised a new member of this superfamily, a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum (R/PMH) both structurally and kinetically. The 1.42 A crystal structure shows structural homology to arylsulfatases with conservation of the core alpha/beta-fold, the mononuclear active site and most of the active-site residues. Sulfatases use a unique formylglycine nucleophile, formed by posttranslational modification of a cysteine/serine embedded in a signature sequence (C/S)XPXR. We provide mass spectrometric and mutational evidence that R/PMH is the first non-sulfatase enzyme shown to use a formylglycine as the catalytic nucleophile. R/PMH hydrolyses phosphonate monoesters and phosphate diesters with similar efficiency. Burst kinetics suggest that substrate hydrolysis proceeds via a double-displacement mechanism. Kinetic characterisation of active-site mutations establishes the catalytic contributions of individual residues. A mechanism for substrate hydrolysis is proposed on the basis of the kinetic data and structural comparisons with E. coli alkaline phosphatase and Pseudomonas aeruginosa arylsulfatase. R/PMH represents a further example of conservation of the overall structure and mechanism within the alkaline phosphatase superfamily.
Jonas
Stefanie
S
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB21GA, UK.
van Loo
Bert
B
Hyvönen
Marko
M
Hollfelder
Florian
F
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D016296:Mutagenesis / Q000187:drug effects; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D016961:Rhizobium leguminosarum / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000187:drug effects
D008670:Metals; D050505:Mutant Proteins; C032736:N-formylglycine; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D005998:Glycine