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 Km and Vmax 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.

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

“…owensensis coAHASL260.1706.50.70 ± 0.0253.78 ± 2.68This study T . maritima AHASL66.4857.0246.00 ± 7.0016.40 ± 2.00 8 Thermus aquaticus AHASNA708.0NA c NA 26 Bacillus sp.AHASNA756.0NANA 26 B . stearothermophilus AHASL63.350NA3.0012.00 ± 2.00 9 E .…”

Optimization of expression and properties of the recombinant acetohydroxyacid synthase of Thermotoga maritima

“… The data provide additional support of the characterization of the biophysical and biochemical properties of the enzyme acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima (Eram et al, 2015) [1] . The genes encoding the enzyme subunits have been cloned and expressed in the mesophilic host Escherichia coli .…”

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

“…The recombinant plasmids for the expression of genes encoding the catalytic and regulatory subunits were constructed and propagated in Escherichia coli strain DH5α. The recombinant proteins overexpressed under the control of T7 polymerase of the plasmid pET30a (+) (Novagen, WI, USA) in E. coli BL21 (DE3) Rosetta 2 [F − ompT hsdS B (rB − mB − ) gal dcm pRARE27 ( CamR )] (Novagen, WI, USA) as described elsewhere [10] , [11] . The recombinant catalytic subunit was purified by heat-induced precipitation of the host proteins (1 h at 80 °C) followed by DEAE and HAP column chromatography as described previously [10] , [11] .…”

“…The AHAS activity was followed during the purification and the purity of the final active fractions were tested by SDS-PAGE. The inactive small subunit was also overexpressed in the same host and purified by denaturing IMAC and on column refolding of the denatured protein using a previously described procedure [10] . The purity of the desired protein in the fractions was checked on SDS-PAGE.…”

“…It has been shown that Thermotoga maritima does not require the addition of any branched chain amino acids (BCAA) to the growth medium for optimal growth, suggesting the organism is equipped for biosynthesis of these compounds [9] . The properties of an anabolic AHAS from the hyperthermophilic bacterium T. maritima has recently been reported [10] . The purified catalytic subunit of the enzyme is FAD- and TPP-dependent and efficiently catalyzes the production of acetolactate from pyruvate with a k cat of about 100 s −1 .…”