R.K. Wierenga

Crystal structure of FadR, a fatty acid‐responsive transcription factor with a novel acyl coenzyme A‐binding fold

FadR is a dimeric acyl coenzyme A (acyl CoA)‐binding protein and transcription factor that regulates the expression of genes encoding fatty acid biosynthetic and degrading enzymes in Escherichia coli. Here, the 2.0 Å crystal structure of full‐length FadR is described, determined using multi‐wavelength anomalous dispersion. The structure reveals a dimer and a two‐domain fold, with DNA‐binding and acyl‐CoA‐binding sites located in an N‐terminal and C‐terminal domain, respectively. The N‐terminal domain contains a winged helix–turn–helix prokaryotic DNA‐binding fold. Comparison with known structures and analysis of mutagenesis data delineated the site of interaction with DNA. The C‐terminal domain has a novel fold, consisting of a seven‐helical bundle with a crossover topology. Careful analysis of the structure, together with mutational and biophysical data, revealed a putative hydrophobic acyl‐CoA‐binding site, buried in the core of the seven‐helical bundle. This structure aids in understanding FadR function at a molecular level, provides the first structural scaffold for the large GntR family of transcription factors, which are keys in the control of metabolism in bacterial pathogens, and could thus be a possible target for novel chemotherapeutic agents.

The crystal structure of delta(3)-delta(2)-enoyl-CoA isomerase.

The active-site geometry of the first crystal structure of a Delta(3)-Delta(2)-enoyl-coenzyme A (CoA) isomerase (the peroxisomal enzyme from the yeast Saccharomyces cerevisiae) shows that only one catalytic base, Glu158, is involved in shuttling the proton from the C2 carbon atom of the substrate, Delta(3)-enoyl-CoA, to the C4 atom of the product, Delta(2)-enoyl-CoA. Site-directed mutagenesis has been performed to confirm that this glutamate residue is essential for catalysis. This Delta(3)-Delta(2)-enoyl-CoA isomerase is a hexameric enzyme, consisting of six identical subunits. It belongs to the hydratase/isomerase superfamily of enzymes which catalyze a wide range of CoA-dependent reactions. The members of the hydratase/ isomerase superfamily have only a low level of sequence identity. Comparison of the crystal structure of the Delta(3)-Delta(2)-enoyl-CoA isomerase with the other structures of this superfamily shows only one region of large structural variability, which is in the second turn of the spiral fold and which is involved in defining the shape of the binding pocket.

Structure determination and refinement at 2.44 A resolution of argininosuccinate lyase from Escherichia coli.

Escherichia coli argininosuccinate lyase has been crystallized from a highly concentrated sample of purified recombinant alpha-methylacyl-CoA racemase, in which it occurred as a minor impurity. The structure has been solved using molecular replacement at 2.44 A resolution. The enzyme is tetrameric, but in this crystal form there is a dimer in the asymmetric unit. The tetramer has four active sites; each active site is constructed from loops of three different subunits. One of these catalytic loops, near residues Ser277 and Ser278, was disordered in previous structures of active lyases, but is very well ordered in this structure in one of the subunits owing to the presence of two phosphate ions in the active-site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex.

Development of MLPA for human ACAT1 gene and identification of a heterozygous Alu-mediated deletion of exons 3 and 4 in a patient with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency

Mitochondrial acetoacetyl-CoA thiolase deficiency is an autosomal recessive disorder, characterized by intermittent ketoacidosis. We developed a multiplex ligation-dependent probe amplification method for mutation detection in the ACAT1 gene, which encodes this enzyme, and validated it using DNAs from two previously reported patients having partial deletion and duplication in this gene. Using this method, we identified a heterozygous deletion including exons 3-4 in a third patient, likely due to Alu-mediated non-equal homologous recombination between Alu sequences.

Crystallization and characterization of the dehydrogenase domain from rat peroxisomal multifunctional enzyme type 1

Peroxisomal multifunctional enzyme type 1 from rat (perMFE-1) is a monomeric multidomain protein shown to have 2-enoyl-CoA hydratase/Delta(3)-Delta(2)-enoyl-CoA isomerase and (3S)-hydroxyacyl-CoA dehydrogenase domains followed by a C-terminal extension of 130 amino acids with unknown function apart from being a carrier of the peroxisomal targeting signal type 1. The truncated perMFE-1 without the N-terminal hydratase/isomerase domain (perMFE-1DH; residues 260-722) was overexpressed as an enzymatically active recombinant protein, purified and characterized. Using (3S)-hydroxydecanoyl-CoA as a substrate, the specific enzymatic activity of perMFE-1DH was determined to be 2.2 micromol min(-1) mg(-1), comparable with that of perMFE-1 purified from rat liver (2.8 micromol min(-1) mg(-1)). The protein was crystallized in the apo form by the hanging-drop method and a complete data set to 2.45 A resolution was collected using a rotating-anode X-ray source. The crystals have primitive tetragonal symmetry, with unit-cell parameters a = b = 125.9, c = 60.2 A.

Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from Mycobacterium smegmatis

An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 Å resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six β-strands forming an anti-parallel β-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.

Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis.

Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The Lα1-covering loop-Lα2 region, together with the Nβ2-Nα2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains.