Cloning, Sequence, and Expression of Kynureninase fromPseudomonas fluorescens

We have cloned the gene encoding kynureninase fromPseudomonas fluorescensusing a restriction site polymerase chain reaction technique (RS-PCR) (G. Sarkar, R. T. Turner, and M. E. BolanderPCR Methods Appl.2, 318–322, 1993) and expressed the enzyme inEscherichia coliDH5α F′. The kynureninase gene has an open reading frame (ORF) of 1251 base pairs that codes for a protein of 416 amino acids with a calculated molecular weight of 45,906. The protein purified fromP. fluorescenshas N-terminal threonine and an observed molecular weight of 45,787 by electrospray mass spectrometry, suggesting that the N-terminal methionine is removed by posttranslational processing. The complete gene was obtained by PCR and inserted into pTZ18U. The resultant plasmid was used to transformE. coliDH5α F′, and these cells overexpressed kynureninase to about 37% of total soluble protein. The isolated recombinant protein has molecular weight andKmvalues identical to those of the native protein fromP. fluorescens.The amino acid sequence exhibits 29% identity with those of rat and human kynureninases and 32% identity with the amino acid sequence translated from aSaccharomyces cerevisiaeORF. Alignment of the four sequences shows a highly conserved region which corresponds to the pyridoxal-5′-phosphate (PLP) binding site of rat kynureninase. Based on this alignment, we predict that Lys227 and Asp212 inP. fluorescenskynureninase are involved in pyridoxal-5′-phosphate binding.P. fluorescenskynure- ninase also exhibits significant homology to thenifS gene product, cysteine desulfurase, and to eucaryotic serine/pyruvate aminotransferases, suggesting that it is a member of subgroup IV of the aminotransferase family of PLP-dependent enzymes.