We assigned these enzymes to group 2. Further analysis revealed several microorganisms (Agrobacterium vitis S4, Bordetella petrii DSM 12804, Vibrio vulnificus YJ016, Sideroxydans lithotrophicus ES-1) whose IDO homologues are expressed only in combination with a specific efflux pump (RhtA/RhtB exporter family) without AR in the same operon regulated by a LysR-type repressor. These dioxygenases were assigned to the third group [Fig. 1 (5, 6, 7)]. By way of

analogy to B. thuringiensis, we proposed that the operons from the learn more first, second and third groups could be involved in the synthesis and excretion of special derivatives of the hydroxylated free l-amino acids produced by their corresponding IDO homologues. In several microbes that we assigned to the fourth group (e.g. Gluconacetobacter diazotrophicus PAl 5 and Proteasome purification Pseudomonas fluorescens Pf0-1), the IDO homologue genes belong to the operons assumed to be involved in the synthetic

process, one stage of which is hydroxylation of an unknown substrate [Fig. 1 (8, 9)]. In some bacteria (e.g. Burkholderia oklahomensis EO147, Burkholderia pseudomallei 668, Photorhabdus luminescens ssp. laumondii TTO1 and Photorhabdus asymbiotica ATCC 43949), the IDO is thought to be co-expressed with polyketide/nonribosomal peptide synthetase-like protein. We proposed that these dioxygenases can be involved in the synthesis of peptide antibiotics containing hydroxylated l-amino acid residues and may also hydroxylate free l-amino acids [Fig. 1 (10)]. We assigned these dioxygenases to the fifth group. Many bacteria encode IDO homologues that are not part of an operon structure and can hydroxylate unpredictable substrates, including free l-amino acids; we included these enzymes in the sixth group. Based on the data obtained thus far, we assumed that the free amino acid dioxygenases were likely to belong to any group except group number four. Eight members of the PF10014 family – IDO (group

1, as a control enzyme); PAA (group 2); AVI, BPE (group 3); PLU (group 5); MFL, GOX and GVI (group 6) – were arbitrarily chosen for cloning and expression in E. coli to examine their substrate specificities with regard to canonical l-amino acids (Table 1). Using standard methods, we expressed selected enzymes as his6-tag proteins and purified them to near homogeneity using conventional PLEKHM2 IMAC. Because our goal was to identify enzymes possessing high hydroxylase activities with potential for biotechnology applications, we first performed a high-throughput analysis for dioxygenase substrate specificity with 20 canonical l-amino acids using TLC analysis of the reaction mixture products (Fig. 2a,b). We found that new amino group-containing substances are formed by hydroxylation reactions with l-isoleucine (IDO, PAA), l-leucine (all enzymes with exception of GVI and PLU), l-methionine (all enzymes, but the activity of PLU was rather low) and l-threonine (BPE, AVI) (Fig. 2c).