Sergey Vasil'evich Smirnov

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.

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a alpha-ketoglutaric acid (alpha-KG) dependent l-Ile dioxygenase (IDO) and requires alpha-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in alpha-KG dependent dioxygenases, is found in RBTH_06809.

Characterization of Bacillus thuringiensis l-Isoleucine Dioxygenase for Production of Useful Amino Acids

ABSTRACT We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst.

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.

Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626.

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM α-ketobutyrate, and 100 mM L-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mM Tris–HCl buffer (pH 7.5) containing 2 mM MnCl2·4H2O at 28 °C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplex AKU 626. Mn2+ and pyridoxal 5′-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.

l-Leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst

Abstractl-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of l-leucine and l-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of l-methionine and l-ethionine in the same manner as previously described l-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.

A novel l‐isoleucine‐4′‐dioxygenase and l‐isoleucine dihydroxylation cascade in Pantoea ananatis

A unique operon structure has been identified in the genomes of several plant‐ and insect‐associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli‐HilA) or both hilA and hilB (E. coli‐HilAB) in the presence of l‐isoleucine resulted in the conversion of l‐isoleucine into two novel biogenic compounds: l‐4′‐isoleucine and l‐4,4′‐dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli‐HilA and E. coli‐HilAB strains: l‐isoleucine, 2‐oxoglutarate: oxygen oxidoreductase (4′‐hydroxylating) (HilA) and l‐4′‐hydroxyisoleucine, 2‐oxoglutarate: oxygen oxidoreductase (4‐hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C‐4(4′)‐hydroxylation of l‐isoleucine in bacteria are also discussed. According to first hypothesis, the l‐isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C‐4(4′)‐hydroxylation of l‐isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)‐furanones and analogs of N‐acyl homoserine lactone.