Leonid Romanovich Ptitsyn

Molecular cloning and characterization of Escherichia coli K12 ygjG gene

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 Kmvalues 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.

The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression

Overexpression of the yeaS gene encoding a protein belonging to the RhtB transporter family conferred upon cells resistance to glycyl‐l‐leucine, leucine analogues, several amino acids and their analogues. yeaS overexpression promoted leucine and, to a lesser extent, methionine and histidine accumulation by the respective producing strains. Our results indicate that yeaS encodes an exporter of leucine and some other structurally unrelated amino acids. The expression of yeaS (renamed leuE for “leucine export”) was induced by leucine, l‐α‐amino‐n‐butyric acid and, to a lesser extent, by several other amino acids. The global regulator Lrp mediated this induction.

Identification of Escherichia coli K12 YdcW protein as a γ-aminobutyraldehyde dehydrogenase

γ‐Aminobutyraldehyde dehydrogenase (ABALDH) from wild‐type E. coli K12 was purified to apparent homogeneity and identified as YdcW by MS‐analysis. YdcW exists as a tetramer of 202 ± 29 kDa in the native state, a molecular mass of one subunit was determined as 51 ± 3 kDa. K m parameters of YdcW for γ‐aminobutyraldehyde, NAD+ and NADP+ were 41 ± 7, 54 ± 10 and 484 ± 72 μM, respectively. YdcW is the unique ABALDH in E. coli K12. A coupling action of E. coli YgjG putrescine transaminase and YdcW dehydrogenase in vitro resulted in conversion of putrescine into γ‐aminobutyric acid.