Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

Abstract

Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å , respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H 2 O) 6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.Keywords: acetohydroxyacid synthase; actolactate synthase; regulatory subunit; ACT domain; AHAS; protein refolding Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes two physiologically significant reactions in the synthesis of isoleucine, valine, and leucine. In the first reaction, which is an initial step in the synthesis of isoleucine, 2-aceto-2-hydroxy butyrate is produced by condensation of 2-ketobutyrate with pyruvate. In the second reaction, 2-acetolactate is synthesized from two pyruvate molecules, and this provides substrates for synthesis of valine and leucine (Umbarger 1978(Umbarger , 1987Chipman et al. 1998). This pathway of branched-chain amino acid biosynthesis is characteristic for bacteria, fungi, algae, and higher plants, but not for animals. Accordingly, AHAS inhibitors have been developed as herbicides which have found broad application (Short and Colborn 1999). The inhibitors of branched-chain amino acids synthesis are also being tested as potential antituberculosis agents (Grandoni et al. 1998;Zohar et al. 2003;Choi et al. 2005).Three different FAD-dependent AHAS isozymes (I, II, and III) are found in enterobacteria (e.g., Escherichia ), but most other organisms from the bacteria encode only a single AHAS enzyme, similar to isozyme III from E. coli. The acetohydroxyacid synthases are multimeric proteins, and most frequently their biological unit is composed of two catalytic subunits (CSU) and two small regulatory subunits (SSU). In the absence of the SSU subunit, the CSU dimer is unstable (Vyazmensky et al. 1996), suggesting that in the holoenzyme the AHAS dimer is stabilized by a SSU dimer. The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al. 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al. 2004), and the reconstituted enzymes form stable heterotetramers. The properly associated holoenzymes show full catalytic activity, and are also susceptible to valine inhibition.Previous mutagenesis studies (Mendel et al. 2001;Kaplun et al. 2006) revealed that the valine binding sites are located at the dimerizatio...

The Crystallization Expert System Xtaldb, and its Application to the Structure of the 5'- nucleotidase YfbR and Other Proteins

“…Eventually, protein purification was carried out under denaturing conditions, by taking the insoluble protein fraction from the cell fractionation spin (see Fig. 3-3) and dissolving in binding buffer plus 6 M guanidine hydrochloride (Petkowski et al, 2007).…”

“…The crystallization of TM0549 was performed in the presence of a relatively high concentration (0.5 M) of L-arginine, which was determined by means of a screen of refolding agents (Petkowski et al, 2007). While the use of L-arginine in preventing aggregation in protein refolding is well known (Tsumoto et al, 2004), the use of the amino acid as an additive for crystallization has not been widely reported.…”

“…50×50×50 µL crystals appeared after about 24 hours. The structure was solved by Se-Met SAD at by Janusz Petkowski (Petkowski et al, 2007).…”

“…TM0549 from Thermotoga maritima is similar by sequence to the so-called small regulatory subunit of the acetohydroxy acid synthase isoform III of E. coli (Kaplun et al, 2006). Upon determination of the crystal structure, the necessity of including Mg formate in the crystallization conditions became obvious, as each monomer contained an ordered Mg(H 2 O) 6 2+ ion that formed major crystal contacts, shown as space-filling spheres in Fig. 3-9 (Petkowski et al, 2007). It is unclear if this metal ion is necessary for the physiological functioning of the protein, as the E. coli ortholog was also crystallized in Mg 2+ but did not contain an ordered ion in the electron density (Kaplun et al, 2006).…”

“…Most importantly, the structures of the uncharacterized proteins solved resulted in more extensive explorations of structure-function relationships. The role of TM0549, as a putative regulatory subunit for acetohydroxyacid synthase was explored (Petkowski et al, 2007). Stuctural studies of TM1030 led to a plausible mechanism of DNA-binding for this member of the TetR transcriptional regulator family .…”