Cloning and Characterization of katA, Encoding the Major Monofunctional Catalase from Xanthomonas campestris pv. phaseoli and Characterization of the Encoded Catalase KatA
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- Chauvatcharin, N., Vattanaviboon, P., Switala, J. et al. Curr Microbiol (2003) 46: 0083. doi:10.1007/s00284-002-3812-8
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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 Km and Vmax 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.