Cloning, sequence, and expression of Kynureninase from Pseudomonas fluorescens
Cloning, sequence, and expression of Kynureninase from Pseudomonas fluorescens
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DOI：
10.1006/abbi.1997.0220
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发表时间：
1997-08-15
影响因子：
3.9
通讯作者：
Phillips, RS
中科院分区：
文献类型：
--
作者：
Koushik, SV;Sundararaju, B;Phillips, RS
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 DH5 alpha 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 DH5 alpha F', and these cells overexpressed kynureninase to about 37% of total soluble protein, The isolated recombinant protein has molecular weight and K-m 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 TV of the aminotransferase family of PLP-dependent enzymes. (C) 1997 Academic Press.
