FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1.
Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component of the biosphere. The crystal structure of this F/10 family xylanase, which has a triosephosphate isomerase (TIM) barrel (β/α)8 fold, has been solved to small‐molecule accuracy at atomic resolution (1.11 Å) at 293 K (RTUX) and at ultrahigh resolution (0.89 Å) at 100 K (CTUX) using X‐ray diffraction data sets collected on a synchrotron light source, resulting in R/Rfree values of 9.94/12.36 and 9.00/10.61% (for all data), respectively. Both structures were refined with anisotropic atomic displacement parameters. The 0.89 Å structure, with 177 476 observed unique reflections, was refined without any stereochemical restraints during the final stages. The salt bridge between Arg124 and Glu232, which is bidentate in RTUX, is water‐mediated in CTUX, suggesting the possibility of plasticity of ion pairs in proteins, with water molecules mediating some of the alternate arrangements. Two buried waters present inside the barrel form hydrogen‐bond interactions with residues in strands β2, β3, β4 and β7 and presumably contribute to structural stability. The availability of accurate structural information at two different temperatures enabled the study of the temperature‐dependent deformations of the TIM‐barrel fold of the xylanase. Analysis of the deviation of corresponding Cα atoms between RTUX and CTUX suggests that the interior β‐strands are less susceptible to changes as a function of temperature than are the α‐helices, which are on the outside of the barrel. βα‐loops, which are longer and contribute residues to the active‐site region, are more flexible than αβ‐loops. The 0.89 Å structure represents one of the highest resolution structures of a protein of such size with one monomer molecule in the asymmetric unit and also represents the highest resolution TIM‐barrel fold structure to date. It may provide a useful template for theoretical modelling studies of the structure and dynamics of the ubiquitous TIM‐barrel fold.
FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284-15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with K(i) values of 41 and 12 nM respectively. A high-resolution (1.35 A; 1 A=0.1 nm) crystal structure of the FAH.CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 mumol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation.
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