Indexed on: 10 Jan '03Published on: 10 Jan '03Published in: Current Microbiology
The first cloning and characterization of the gene katA, encoding the major catalase (KatA), from Xanthomonas is reported. A reverse genetic approach using a synthesized katA-specific DNA probe to screen a X. campestris pv. phaseoli genomic library was employed. A positively hybridizing clone designated pKat29 that contained a full-length katA was isolated. Analysis of the nucleotide sequence revealed an open reading frame of 1,521 bp encoding a 507-amino acid protein with a theoretical molecular mass of 56 kDa. The deduced amino acid sequence of KatA revealed 84% and 78% identity to CatF of Pseudomonas syringae and KatB of P. aeruginosa, respectively. Phylogenetic analysis places Xanthomonas katA in the clade I group of bacterial catalases. Unexpectedly, expression of katA in a heterologous Escherichia coli host resulted in a temperature-sensitive expression. The KatA enzyme was purified from an overproducing mutant of X. campestris and was characterized. It has apparent K(m) and V(max) values of 75 m M [H(2)O(2)] and 2.55 x 10(5) micromol H(2)O(2) micromol heme(-1) s(-1), respectively. The enzyme is highly sensitive to 3-amino-1,2,4-triazole and NaN(3), has a narrower optimal pH range than other catalases, and is more sensitive to heat inactivation.