Single-site mutations on the catalase-peroxidase from Sinorhizobium meliloti: role of the distal Gly and the three amino acids of the putative intrinsic cofactor.
KatB is the only catalase-peroxidase identified so far in Sinorhizobium meliloti. It plays a housekeeping role, as it is expressed throughout all the growth phases of the free-living bacterium and also during symbiosis. This paper describes the functional and structural characterization of the KatB mutants Gly303Ser, Trp95Ala, Trp95Phe, Tyr217Leu, Tyr217Phe and Met243Val carried out by optical and electron spin resonance spectroscopy. The aim of this work was to investigate the involvement of these residues in the catalatic and/or peroxidatic reaction and falls in the frame of the open dispute around the factors that influence the balance between catalatic and peroxidatic activity in heme enzymes. The Gly303 residue is not conserved in any other protein of this family, whereas the Trp95, Tyr217 and Met243 residues are thought to form an intrinsic cofactor that is likely to play a role in intramolecular electron transfer. Spectroscopic investigations show that the Gly303Ser mutant is almost similar to the wild-type KatB and should not be involved in substrate binding. Mutations on Trp95, Tyr217 and Met243 clear out the catalatic activity completely, whereas the peroxidatic activity is maintained or even increased with respect to that of the wild-type enzyme. The k (cat) values obtained for these mutants suggest that Trp95 and Tyr217 form a huge delocalized system that provides a pathway for electron transfer to the heme. Conversely, Met243 is likely to be placed close to the binding site of the organic molecules and plays a crucial role in substrate docking.
Ardissone
Silvia
S
Dipartimento di Chimica I. F. M., Università di Torino, via Pietro Giuria 7, 10125, Turin, Italy.
Laurenti
Enzo
E
Frendo
Pierre
P
Ghibaudi
Elena M
EM
Puppo
Alain
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D002374:Catalase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D000596:Amino Acids; D001386:Azides; D003486:Cyanides; D004791:Enzyme Inhibitors; D010544:Peroxidases; D002374:Catalase