Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

Abstract

Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent K_m and V_max for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far.

Keywords: AHAS, acetohydroxyacid synthase; Acetohydroxyacid synthase; BCAA, branched chain amino acid; Branched-chain amino acids; CCE, crude cell extract; CFE, cell-free extract; HTCCE, heat-treated crude cell extract; Hyperthermophiles; IB, inclusion body; IMAC, immobilized metal affinity chromatography; TPP, thiamine pyrophosphate; Thermotogales; TmAHAS, Thermotoga maritima acetohydroxyacid synthase; ilv, isoleucine, leucine, valine.

Graphical abstract

Fig. 1

Biosynthesis pathways of branched chain amino acids and the butanediol pathway (Boxed). TD, threonine deaminase (EC 4.3.1.19); KARI, ketol-acid reductoisomerase (EC 1.1.1.86); DHAD, dihydroxyacid dehydratase (EC 4.2.1.9) ; TA, transaminase (EC 2.6.1.42, EC 2.6.1.66, EC 2.6.1.6); IPMS, 2-isopropylmalate synthase (EC 2.3.3.13); IPMD, isopropylmalate dehydratase (EC 4.2.1.33); IPMDH, 3-isopropylmalate dehydrogenase (EC 1.1.1.85); AD, acetolactate decarboxylase (EC 4.1.1.5); AR, acetoin reductase (EC 1.1.1.4).

Fig. 2

Gene organization of the ilv gene cluster in T. maritima. The filled arrows indicate the genes encoding enzymes involved in BCAA biosynthesis. The black arrows are the hypothetical genes encoding the catalytic and regulatory subunits of AHAS. Abbreviations: ilvB: catalytic subunit of acetohydroxyacid synthase (AHASL); ilvN, regulatory subunit of acetohydroxyacid synthase (AHASS); ilvC, ketol-acid reductoisomerase (KARI); ilvD ; dihydroxy-acid dehydratase (DHAD); leuA, 2-isopropylmalate synthase (IPMS); leuC 3-isopropylmalate dehydratase large subunit (IPMDL); and leuD, 3-isopropylmalate dehydratase small subunit (IPMDS).

Fig. 3

SDS-PAGE analysis of the purified proteins. (A) The catalytic subunit on 12.5% SDS-PAGE and with lane M, pre-stained protein ladder, Lane 1, 2 µg of the purified protein; lane 2, 4 µg of the purified protein. (B) The regulatory subunit of TmAHAS on 15% SDS-PAGE with Lane 1, 8 µg of the purified protein; lane 2, 4 µg of the purified protein; lane M, pre-stained protein ladder.

Fig. 4

Effect of pH and temperature on AHAS activity. (A) To determine the optimal pH, the AHAS activity was determined in ● sodium acetate buffer (pH values 4.0, 5.0, and 5.5), ○ sodium phosphate buffer (pH 6.2, 7.0, 7.5, and 8.0), ▼ EPPS (pH 8.0, 8.4, 9.0, and 9.5), ∆ glycine (pH 9.5 and 10), and ■ CAPS (pH 10.5 and 11.0). The relative activities of 100% equals to the highest measured specific activity (114 U/mg) at 80 ºC in sodium phosphate buffer, pH 7.0. (B) To determine the temperature dependence, the reaction vials were incubated at each temperature for 4 min before starting the reaction by adding the enzyme. The inset shows the Arrhenius plot based on the linear part of the plot B (temperatures 60–85 ºC). The relative activity of 100% equals to the highest measured specific activity (235 U/mg) at 85 ºC.

Fig. 5

Conserved motifs in AHASs. The highly conserved motifs in AHASs are boxed: (A) The FAD-binding motif (boxed). (B) and TPP-binding motif (boxed). The AHAS amino acid sequences of different thermophilic and mesophilic organisms were aligned using MegAlign software (Lasergene, DNAStar, Madison, USA).