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

Abstract

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.

Industrial Application of 2-Oxoglutarate-Dependent Oxygenases

“…In the native organism a second 2OGX, named HilB, further processes 4'-hydroxyisoleucine into dihydroxylated 4,4'-dihydroxyisoleucine (Figure 4). In another study, Smirnov et al investigated the substrate scope of HilA and showed promiscuous activity for the conversion of L-valine into L-4-hydroxyvaline [70].…”

“…In contrast to the C4 selectivity of IDO, the 2-oxoglutarate-dependent oxygenase HilA from Pantoea ananatis hydroxylates L-isoleucine on the C3 methyl group to produce 2-amino-3-(hydroxymethyl)pentanoic acid or 4 -hydroxyisoleucine [69,70]. In the native organism a second 2OGX, named HilB, further processes 4 -hydroxyisoleucine into dihydroxylated 4,4 -dihydroxyisoleucine (Figure 4).…”

Biochemical Diversity of 2-Oxoglutarate-Dependent Oxygenases

“…243 The products of two adjacent genes in Pantoea ananatis, HilA and HilB, catalyse sequential hydroxylation reactions at the 4′ and 4 positions, respectively, of l-Ile ( Figure 1.9H). 244 Related family members were identified in a range of other bacteria, in several cases using l-Leu as the preferred substrate and yielding 4-hydroxyleucine (Figure 1.9I). 245 Alternatively, l-Leu 5-hydroxylase (LdoA, Figure 1.9J) was characterized from Nostoc punctiforme.…”

“…246 All of these enzymes also act on other substrates with reduced catalytic efficiencies (including Leu/Ile substitution), in various cases forming Met sulfoxide, 4-hydroxyvaline and 4-hydroxythreonine (not shown). [243][244][245] Four other enzymes are mentioned in this section because they modify amino acid-like substrates or combine another reaction with amino acid hydroxylation. SadA from Burkholderia ambifaria acts on several N-substituted amino acids with hydrophobic side chains, notably catalysing 3R hydroxylation of N-succinyl-l-Leu (Figure 1.10A).…”

A Multi-Enzymatic Cascade Reaction for the Stereoselective Production of γ-Oxyfunctionalyzed Amino Acids

“…Natural amino acid hydroxylation is carried out by Fe(II)/α-ketoglutarate dependent enzymes. One of the most promising is the L-isoleucine dioxygenase from Bacillus thuringiensis (BtDO), which catalyzes the enantioselective hydroxylation of several hydrophobic amino acids with specificity for the γ-position (Kodera et al, 2009 ; Hibi et al, 2011 ; Ogawa et al, 2011 ; Smirnov et al, 2013 ). Interestingly, BtDO also catalyzes the highly enantioselective sulfoxidation of L-methionine (Scheme 1 ; Hibi et al, 2013b ).…”