Cloning, sequence, and expression of kynureninase from Pseudomonas fluorescens.
We have cloned the gene encoding kynureninase from Pseudomonas fluorescens using a restriction site polymerase chain reaction technique (RS-PCR) (G. Sarkar, R. T. Turner, and M. E. Bolander PCR Methods Appl. 2, 318-322, 1993) and expressed the enzyme in Escherichia coli DH5a 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 from P. fluorescens has 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 transform E. coli DH5alpha F', and these cells overexpressed kynureninase to about 37% of total soluble protein. The isolated recombinant protein has molecular weight and Km values identical to those of the native protein from P. 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 a Saccharomyces cerevisiae ORF. 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 in P. fluorescens kynureninase are involved in pyridoxal-5'-phosphate binding. P. fluorescens kynureninase also exhibits significant homology to the nifS 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.
Koushik
S V
SV
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Sundararaju
B
B
McGraw
R A
RA
Phillips
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D011994:Recombinant Proteins; D006867:Hydrolases; C021480:kynureninase