l-methionine, l-leucine, l-isoleucine and l-threonine were found to be catalysed by the investigated enzymes, producing l-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family Ixazomib manufacturer for which free l-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed. Hydroxylation of
free canonical l-amino acids is an interesting and growing field of biotechnology and molecular biology research. Introduction of a functional hydroxyl group into amino acid molecules makes possible synthesis of fine chemicals (Blaskovich et al., 1998). In addition, the hydroxylated amino acid may itself be biologically active with pharmacological significance (Jette et al., 2009) and/or may be involved in bacterial metabolic regulation (Ogawa et al., 2011). In bacteria, the hydroxylation of free l-amino acids
NVP-BKM120 chemical structure is usually catalysed by specific Fe(II)/α-ketoglutarate-dependent dioxygenases (Hausinger, 2004). Because both l-amino acids and α-ketoglutarate are involved in cell metabolism, metabolic engineering of Escherichia coli could be used for the microbiological production of target hydroxylated l-amino acids (Shibasaki Bumetanide et al., 2000; Smirnov et al., 2010; Ogawa et al., 2011).[ Correction added after online publication 17 April 2012: Kim et al., 2010 and Smirnov et al., 2010 references swapped throughout ]. Thus, identification of novel l-amino acid dioxygenases or l-amino acid hydroxylation activities may facilitate industrial bioprocesses
to produce novel pharmaceuticals and synthons for organic chemistry. In addition, understanding the hydroxylation of free l-amino acids could facilitate the discovery of novel biosynthetic processes and regulatory mechanisms in bacteria. Recently, we described the cloning and characterization of l-isoleucine-4-hydroxylase (IDO) from Bacillus thuringiensis (Kodera et al., 2009; Smirnov et al., 2010; Hibi et al., 2011; Ogawa et al., 2011). IDO hydroxylated several hydrophobic aliphatic l-amino acids, including l-leucine, and generated l-methionine sulfoxide from l-methionine. In this work, we used IDO homologues from several bacteria to examine the substrate specificities of novel dioxygenases in regard to other canonical l-amino acids and to determine kinetic constants for l-isoleucine, l-leucine and l-methionine. To construct the pET-HT-IDO, pET-HT-PAA, pET-HT-MFL and pET-HT-GOX plasmids, the following DNA fragments were amplified: (1) a 776-bp ‘IDO’ fragment, using the primers svs 335 (5′-TATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCAAAATGAGTGGCTTTAGCATAGAAGA-3′) and svs 336 (5′-CAGCCGGATCCTTATTTTGTCTCCTTATAA-3′) and the pEL-IDO plasmid (Smirnov et al.