Glycine oxidase from Bacillus subtilis: role of histidine 244 and methionine 261.
The reactions of several mutants at position 244 and 261 of bacterial glycine oxidase (GO) were studied by stopped-flow and steady-state kinetic methods. Substituting H244 with phenylalanine, glutamate, and glutamine and M261 with histidine and tyrosine did not affect the expression of GO and the physicochemical properties of bound FAD. All the H244 and M261 mutants of GO we prepared retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key elements in glycine and sarcosine oxidation. We demonstrated that the substitution of H244 significantly affected the rate of flavin reduction with glycine even if this change did not modify the turnover number, which is frequently increased compared to wild-type GO. However, substitution of M261 affected the interaction with substrates/inhibitors and the rate of flavin reduction with sarcosine and resulted in a decrease in turnover number and efficiency with all the substrates tested. The considerable decrease in the rate of flavin reduction changed the conditions such that it was partially rate-limiting in the catalytic cycle compared to the wild-type GO. Our studies show some similarities, but also major differences, in the catalytic mechanism of GO and other flavooxidases also active on glycine and sarcosine and give insight into the mode of modulation of catalysis and substrate specificities.
Boselli
Angelo
A
Department of Biotechnology and Molecular Sciences, University of Insubria, via JH Dunant 3, 21100, Varese, Italy.
Rosini
Elena
E
Marcone
Giorgia Letizia
GL
Sacchi
Silvia
S
Motteran
Laura
L
Pilone
Mirella S
MS
Pollegioni
Loredano
L
Molla
Gianluca
G
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D008024:Ligands; D050505:Mutant Proteins; D006639:Histidine; D008715:Methionine; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase