Abstract
BackgroundPutrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source. Transaminase and dehydrogenase enzymes seem to be implicated in the degradative pathway of putrescine, in which this compound is converted into γ-aminobutyrate. But genes coding for these enzymes have not been identified so far.ResultsThe 1.8-kbp DNA fragment containing E. coli K12 ygjG gene with aer-ygjG intergenic region was examined. It was found that the fragment contains σ54-depended open reading frame (ORF) of 1,380 nucleotides encoding a 459-amino acid polypeptide of approximately 49.6 kDa. The cytidine (C) residue localized 10 bp downstream of the σ54 promoter sequence was identified as the first mRNA base. The UUG translation initiation codon is situated 36 nucleotides downstream of the mRNA start. The YgjG was expressed as a his6-tag fused protein and purified to homogeneity. The protein catalyzed putrescine:2-oxoglutaric acid (2-OG) aminotransferase reaction (PATase, EC 2.6.1.29). The Km values for putrescine and 2-OG were found to be 9.2 mM and 19.0 mM, respectively. The recombinant enzyme also was able to transaminate cadaverine and, in lower extent, spermidine, and gave maximum activity at pH 9.0.ConclusionExpression of E. coli K12 ygjG coding region revealed σ54-depended ORF which encodes a 459-amino acid protein with putrescine:2-OG aminotransferase activity. The enzyme also was able to transaminate cadaverine and, in lower extent, spermidine.
Highlights
Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source
For more detail characterization of the role of the 5'-flanking region in the ygjG expression and nitrogen starvation induction we compared the expression levels of three different ygjG upstream regions fused to the lacZ reporter gene in plasmid pBRP [23] under different growth conditions. pBR-σ54lacZ-1 and pBR-σ54-lacZ-2 plasmids carried 343-bp KpnIAccIII and 56-bp BglI-AccIII fragments, respectively, containing predicted σ54 promoter sequence, and pBR-lacZ-3 plasmid harbored 282-bp KpnI-BglI fragment upstream of σ54 promoter
We have found that E. coli K-12 ygjG gene encodes for enzyme catalyzing PATase reaction
Summary
Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source. Genes coding for these enzymes have not been identified so far. Polyamines, such as putrescine and spermidine, are present in virtually all living cells, from bacteria to plant and human cells, with a very important though poorly understood biological role [1,2]. They can bind to nucleic acids, stabilize membrane and stimulate activity of several enzymes [2,3,4,5].
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