Cloning and Characterization of katA, Encoding the Major Monofunctional Catalase from Xanthomonas campestris pv. phaseoli and Characterization of the Encoded Catalase KatA

Abstract

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 mM [H₂O₂] and 2.55 × 10⁵ μmol H₂O₂ μmol heme⁻¹ s⁻¹, respectively. The enzyme is highly sensitive to 3-amino-1,2,4-triazole and NaN₃, has a narrower optimal pH range than other catalases, and is more sensitive to heat inactivation.