Molecular cloning and characterization of Escherichia coli K12 ygjG gene.
BACKGROUND
Putrescine 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 gamma-aminobutyrate. But genes coding for these enzymes have not been identified so far.


RESULTS
The 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 sigma54-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 sigma54 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.


CONCLUSIONS
Expression of E. coli K12 ygjG coding region revealed sigma54-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.
Samsonova
Natalya N
NN
Ajinomoto-Genetika Research Institute, 1st Dorozhny pr. 1, Moscow, 113545, Russia. nsams@genetika.ru
Smirnov
Sergey V
SV
Altman
Irina B
IB
Ptitsyn
Leonid R
LR
D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D048168:Escherichia coli K12 / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D023061:Gene Order / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011700:Putrescine / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism; D024363:Transcription Initiation Site
D017931:DNA Primers; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000637:Transaminases; C507750:YgjG protein, E coli; C048018:diamine aminotransferase; D011700:Putrescine