Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus.
Recently, cupin type phosphoglucose isomerases have been described as a novel protein family representing a separate lineage in the evolution of phosphoglucose isomerases. The importance of eight active site residues completely conserved within the cPGI family has been assessed by site-directed mutagenesis using the cPGI from Archaeoglobus fulgidus (AfcPGI) as a model. The mutants T63A, G79A, G79L, H80A, H80D, H82A, E93A, E93D, Y95F, Y95K, H136A, and Y160F were constructed, purified, and the impact of the respective mutation on catalysis and/or metal ion binding as well as thermostability was analyzed. The variants G79A, G79L, and Y95F exhibited a lower thermostability. The catalytic efficiency of the enzyme was reduced by more than 100-fold in the G79A, G79L, H80A, H80D, E93D, Y95F variants and more than 15-fold in the T63A, H82A, Y95K, Y160F variants, but remained about the same in the H136A variant at Ni2+ saturating conditions. Further, the Ni2+ content of the mutants H80A, H80D, H82A, E93A, E93D and their apparent Ni2+ binding ability was reduced, resulting in an almost complete loss of activity and thus underlining the crucial role of the metal ion for catalysis. Evidence is presented that H80, H82 and E93 play an additional role in catalysis besides metal ion binding. E93 appears to be the key catalytic residue of AfcPGI, as the E93A mutant did not show any catalytic activity at all.
Hansen
Thomas
T
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Germany.
Schlichting
Bettina
B
Grötzinger
Joachim
J
Swan
Michael K
MK
Davies
Christopher
C
Schönheit
Peter
P
D019943:Amino Acid Substitution; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009532:Nickel / Q000737:chemistry
D009532:Nickel; D005956:Glucose-6-Phosphate Isomerase