An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis

Abstract

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.

Keywords: amino acid; aminotransferase; archaea; biosynthesis; enzyme; metabolism; ornithine; proline; thermophile; transamination.

Figure 1.

Purification of the TK2101 protein. Three micrograms of the purified TK2101 protein was applied to SDS-PAGE and stained with Coomassie Brilliant Blue (lane 1). M, molecular mass marker.

Figure 2.

Enzymatic properties of the TK2101 protein. A, effect of pH on the TK2101 protein activity. Filled circles, squares, triangles, and diamonds represent the activities in MES (pH 5.5–7.0), HEPES (pH 7.0–8.0), Tricine (pH 8.0–9.0), and CHES (pH 9.0–10.0), respectively. B, effect of temperature on the TK2101 protein activity. C, Arrhenius plot of the data shown in B. Only filled circles were used to estimate the activation energy of the reaction.

Figure 3.

Kinetic studies on the TK2101 protein. A, initial velocities of the aminotransferase reactions with varying concentrations of l-Orn and l-Lys in the presence of 5 mm 2-oxoglutarate. Filled circles and open circles represent initial velocities with l-Orn and l-Lys, respectively. B, initial velocities of the aminotransferase reactions with varying concentrations of 2-oxoglutarate and 2-oxoadipate in the presence of 5 mm l-Orn. Filled squares and open squares represent initial velocities with 2-oxoglutarate and 2-oxoadipate, respectively.

Figure 4.

Putative biosynthesis pathway for Pro in T. kodakarensis. It is expected that l-Pro can be synthesized from l-Orn through three steps containing one non-enzymatic reaction. Possible routes for l-Orn biosynthesis are also shown.

Figure 5.

Growth properties of the parent and the TK2101 gene disruption strain in synthetic amino acid medium. A, growth comparison of the parent and ΔTK2101 strains in the absence of l-Pro and the effects of adding either l-Pro or l-Orn. B, effect of adding l-Orn in the presence of l-Pro. Each value is an average of those from three independent growth experiments. The vertical axis is represented in logarithmic scale. Filled and open symbols represent the parent strain (P) and ΔTK2101 (D), respectively. All media are based on ASW-AA-S⁰-Pyr-Ura-W. Strains and the presence or absence of l-Orn or l-Pro are indicated on the right side of the growth curves.

Figure 6.

Sequence alignment of the TK2101 protein and previously described Orn AT proteins. The circles indicate Lys-292, to which the PLP cofactor is bound, and Glu-235 and Asp-248, which interact with the pyridine ring, based on the structure of the protein from H. sapiens. The nucleotide-binding motif conserved in ω-aminotransferases (Gly-268–Ile–Gly–Arg–Thr–Gly-273) is indicated by a thick bar. Conserved residues are indicated with asterisks. Abbreviations: Bsu, B. subtilis; Hsa, H. sapiens; Sce, S. cerevisiae; Tko, T. kodakarensis.

Figure 7.

Phylogenetic trees of the class III aminotransferase sequences. A, tree including various class III aminotransferase sequences from all three domains, including Orn-AT from eukaryotes and bacteria. B, detailed tree of the clade containing TK1211 and TK2101, which was constructed with only aminotransferase sequences from archaea. Bootstrap values above 50 are shown. In both trees, the TK2101 (star) and TK1211 (circle) sequences are highlighted. Abbreviation of enzymes are as follows: Acorn AT, acetylornithine aminotransferase (Eukaryotes); AGXT2, alanine-glyoxylate aminotransferase; AGXT2L1, ethanolamine–phosphate phospho-lyase; AGXT2L2, 5-phosphohydroxy-l-lysine phospho-lyase; ArgD, acetylornithine aminotransferase (Bacteria); AstC, succinylornithine transaminase; BIO3, adenosylmethionine-8-amino-7-oxononanoate aminotransferase (Eukaryotes); BioA, adenosylmethionine-8-amino-7-oxononanoate aminotransferase (Bacteria); BioK, l-lysine-8-amino-7-oxononanoate transaminase; DavT, 5-aminovalerate aminotransferase; GABA AT, 4-aminobutyrate aminotransferase; Lys AT, l-lysine-ϵ-aminotransferase; LysJ, (LysW)-aminoadipate semialdehyde transaminase; Orn AT, ornithine aminotransferase; PatA, putrescine aminotransferase. Abbreviation of organisms: Aae, Acidiplasma aeolicum; Abo, Aciduliprofundum boonei; Aho, Acidianus hospitalis; Ami, Actinosynnema mirum; Ani, Aspergillus nidulans; Ano, Aspergillus nomius; Asa, Acidilobus saccharovorans; Ath, Arabidopsis thaliana; Bba, Bacillus bataviensis; Bsu, B. subtilis; Cdi, Cuniculiplasma divulgatum; Cgl, Corynebacterium glutamicum; Cla, Caldisphaera lagunensis; Cma, Caldivirga maquilingensis; Dfa, Dictyostelium fasciculatum; Dgr, Deinococcus grandis; Dka, Desulfurococcus kamchatkensis; Dmu, Desulfurococcus mucosus; Eco, E. coli; Hlc, Candidatus heimdallarchaeota archaeon LC_2; Hsa, H. sapiens; Hvo, Haloferax volcanii; Mba, Marine Group III euryarchaeote CG-Bathy1; Mja, Methanocaldococcus jannaschii; Mli, Methanofollis liminatans; Mpa, Mycobacterium paratuberculosi; Mtu, Mycobacterium tuberculosis; Nfa, Nocardia farcinica; Nma, Natrialba magadii; Npe, Natrinema pellirubrum; Nti, Natronorubrum tibetense; Pab, P. abyssi; Pac, Peptoclostridium acidaminophilum; Pau, Paenarthrobacter aurescens; Pde, Pyrodictium delaneyi; Pfl, Pseudomonas fluorescens; Pfu, P. furiosus; Pho, P. horikoshii; Pna, Pyrococcus sp. strain NA2; Poc, Pyrodictium occultum; Ppa, Palaeococcus pacificus; Ppu, Pseudomonas putida; Pst, Pyrococcus sp. ST04; Pto, Picrophilus torridus; Pya, Pyrococcus yayanosii; Rno, Rattus norvegicus; Sac, Sulfolobus acidocaldarius; Sau, Staphylococcus aureus; Sca, Staphylococcus carnosus; Sce, S. cerevisiae; Scl, Streptomyces clavuligerus; Ser, S. erythraea; Sgr, Streptomyces griseus; She, Staphylothermus hellenicus; Sma, Staphylothermus marinus; Sme, Sinorhizobium meliloti; Son, Shewanella oneidensis; Ssc, Sus scrofa; Sso, Sulfolobus solfataricus; Sto, Sulfolobus tokodaii; Sty, Salmonella typhimurium; Tad, Thermofilum adornatus; Tag, Thermosphaera aggregans; Taq, Thermus aquaticus; Tba, Thermococcus barophilus; Tcc, Thermococcus celericrescens; Tct, Thermococcus thioreducens; Teu, Thermococcus eurythermalis; Tfu, Thermofilum uzonense; Tgc, Thermogladius cellulolyticus; Tko, T. kodakarensis; Tli, Thermococcus litoralis; Tpl, Thermoplasmatales archaeon I-plasma; Tpu, Thermoproteus uzoniensis; Tro, Terriglobus roseus; Tsc, Thermus scotoductus; Tsi, Thermococcus sibiricus; Ttp, Thermus thermophiles; Vdi, Vulcanisaeta distributa; Vmo, Vulcanisaeta moutnovskia.