Triosephosphate isomerase: a highly evolved biocatalyst.

Triosephosphate isomerase (TIM) is a perfectly evolved enzyme which very fast interconverts dihydroxyacetone phosphate and D: -glyceraldehyde-3-phosphate. Its catalytic site is at the dimer interface, but the four catalytic residues, Asn11, Lys13, His95 and Glu167, are from the same subunit. Glu167 is the catalytic base. An important feature of the TIM active site is the concerted closure of loop-6 and loop-7 on ligand binding, shielding the catalytic site from bulk solvent. The buried active site stabilises the enediolate intermediate. The catalytic residue Glu167 is at the beginning of loop-6. On closure of loop-6, the Glu167 carboxylate moiety moves approximately 2 Å to the substrate. The dynamic properties of the Glu167 side chain in the enzyme substrate complex are a key feature of the proton shuttling mechanism. Two proton shuttling mechanisms, the classical and the criss-cross mechanism, are responsible for the interconversion of the substrates of this enolising enzyme.

Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.

The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.

Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 A resolution.

beta-Oxidation of amino acyl coenzyme A (acyl-CoA) species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. MFE-2 has a modular organization of three domains. The function of the C-terminal domain of the mammalian MFE-2, which shows similarity with sterol carrier protein type 2 (SCP-2), is unclear. Here, the structure of the SCP-2-like domain comprising amino acid residues 618-736 of human MFE-2 (d Delta h Delta SCP-2L) was solved at 1.75 A resolution in complex with Triton X-100, an analog of a lipid molecule. This is the first reported structure of an MFE-2 domain. The d Delta h Delta SCP-2L has an alpha/beta-fold consisting of five beta-strands and five alpha-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of d Delta h Delta SCP-2L shows a hydrophobic tunnel that traverses the protein, which is occupied by an ordered Triton X-100 molecule. The tunnel is large enough to accommodate molecules such as straight-chain and branched-chain fatty acyl-CoAs and bile acid intermediates. Large empty apolar cavities are observed near the exit of the tunnel and between the helices C and D. In addition, the C-terminal peroxisomal targeting signal is ordered in the structure and solvent-exposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism.

Structural determinants for ligand binding and catalysis of triosephosphate isomerase.

The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate D-glyceraldehyde-3-phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM-IPP and the TIM-PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding.

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.

The TIM-barrel fold: a versatile framework for efficient enzymes.

Recent studies on triosephosphate isomerase (TIM)-barrel enzymes highlight the remarkable versatility of the TIM-barrel scaffold. At least 15 distinct enzyme families use this framework to generate the appropriate active site geometry, always at the C-terminal end of the eight parallel beta-strands of the barrel. Sequence and structure comparisons now suggest that many of the TIM-barrel enzymes are evolutionarily related. Common structural properties of TIM-barrel enzymes are discussed.

Modeling, mutagenesis, and structural studies on the fully conserved phosphate-binding loop (loop 8) of triosephosphate isomerase: toward a new substrate specificity.

Loop 8 (residues 232-242) in triosephosphate isomerase (TIM) is a highly conserved loop that forms a tight binding pocket for the phosphate moiety of the substrate. Its sequence includes the fully conserved, solvent-exposed Leu238. The tight phosphate-binding pocket explains the high substrate specificity of TIM being limited to the in vivo substrates dihydroxyacetone-phosphate and D-glyceraldehyde-3-phosphate. Here we use the monomeric variant of trypanosomal TIM for exploring the structural consequences of shortening this loop. The mutagenesis, guided by extensive modeling calculations and followed up by crystallographic characterization, is aimed at widening the phosphate-binding pocket and, consequently, changing the substrate specificity. Two new variants were characterized. The crystal structures of these variants indicate that in monomeric forms of TIM, the Leu238 side-chain is nicely buried in a hydrophobic cluster. Monomeric forms of wild-type dimeric TIM are known to exist transiently as folding intermediates; our structural analysis suggests that in this monomeric form, Leu238 of loop 8 also adopts this completely buried conformation, which explains its full conservation across the evolution. The much wider phosphate-binding pocket of the new variant allows for the development of a new TIM variant with a different substrate specificity.

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 A 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.

Crystallization and X-ray diffraction analysis of peroxisomal Delta3-Delta2-enoyl-CoA isomerase from Saccharomyces cerevisiae.

The purification, crystallization and X-ray diffraction analysis of Saccharomyces cerevisiae Delta(3)-Delta(2)-enoyl-CoA isomerase is described. Delta(3)-Delta(2)-Enoyl-CoA isomerase is a member of the hydratase/isomerase protein family and is an auxiliary enzyme required for the beta-oxidation of unsaturated fatty acids. It is a hexameric enzyme consisting of six identical 32 kDa subunits of 280 residues each. In crystallization trials three crystal forms were obtained, with tetragonal and hexagonal lattices. A 2.5 A data set was collected from the unliganded hexagonal crystals with an R(merge) of 6.6%. The crystal, with unit-cell parameters a = 116.0, b = 116.0, c = 122.9 A, is likely to have P6(3)22 symmetry.

Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution.

In this paper, we describe the structure of chitinase B from Serratia marcescens, which consists of a catalytic domain with a TIM-barrel fold and a 49-residue C-terminal chitin-binding domain. This chitinase is the first structure of a bacterial exochitinase, and it represents one of only a few examples of a glycosyl hydrolase structure having interacting catalytic and substrate-binding domains. The chitin-binding domain has exposed aromatic residues that contribute to a 55-A long continuous aromatic stretch extending into the active site. Binding of chitin oligomers is blocked beyond the -3 subsite, which explains why the enzyme has chitotriosidase activity and degrades the chitin chain from the nonreducing end. Comparison of the chitinase B structure with that of chitinase A explains why these enzymes act synergistically in the degradation of chitin.

Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase.

Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.

The ionization of a buried glutamic acid is thermodynamically linked to the stability of Leishmania mexicana triose phosphate isomerase.

The amino acid sequence of Leishmania mexicana triose phosphate isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride-induced unfolding. The crystal structure of E65Q was determined. Three important observations were made: (a) there are no structural rearrangements as the result of the substitution; (b) the mutant is more stable than the wild-type; and (c) the stability of the wild-type enzyme shows strong pH dependence, which can be attributed to the ionization of Glu65. Burying of the Glu65 side chain in the uncharged environment of the dimer interface results in a shift in pKa of more than 3 units. The pH-dependent decrease in overall stability is due to weakening of the monomer-monomer interactions (in the dimer). The E65Q substitution causes an increase in stability as the result of the formation of an additional hydrogen bond in each subunit (DeltaDeltaG degrees of 2 kcal.mol-1 per monomer) and the elimination of a charged group in the dimer interface (DeltaDeltaG degrees of at least 9 kcal.mol-1 per dimer). The computated shift in pKa and the stability of the dimer calculated from the charge distribution in the protein structure agree closely with the experimental results. The guanidinium chloride dependence of the unfolding constant was smaller than expected from studies involving monomeric model proteins. No intermediates could be identified in the unfolding equilibrium by combining fluorescence and CD measurements. Study of a stable monomeric triose phosphate isomerase variant confirmed that the phenomenon persists in the monomer.

Crystallization and X-ray diffraction studies of the fatty-acid responsive transcription factor FadR from Escherichia coli.

FadR, an acylCoA-dependent Escherichia coli transcription factor controlling the expression of genes involved in fatty-acid degradation and synthesis, has been crystallized. Crystals of two binary complexes were obtained. The FadR-CoA complex crystallized in space group C222(1), with unit-cell parameters a = 61.3, b = 102.0, c = 91.3 A. The FadR-octanoyl-CoA complex crystallized in space group P6(5)22, with unit-cell parameters a = b = 59.7, c = 296.2 A. Both crystal forms diffracted to 3.5 A on a rotating-anode generator. In both crystal forms, the asymmetric unit contains one subunit. The protein is known to be a homodimer; each subunit consists of two domains of unknown fold. For the acyl-CoA-binding domain, a previously undetected sequence homology to PAS domains, in particular the photoactive yellow protein, is reported.

The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures.

The molecular mechanisms that evolution has been employing to adapt to environmental temperatures are poorly understood. To gain some further insight into this subject we solved the crystal structure of triosephosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima (TmTIM). The enzyme is a tetramer, assembled as a dimer of dimers, suggesting that the tetrameric wild-type phosphoglycerate kinase PGK-TIM fusion protein consists of a core of two TIM dimers covalently linked to 4 PGK units. The crystal structure of TmTIM represents the most thermostable TIM presently known in its 3D-structure. It adds to a series of nine known TIM structures from a wide variety of organisms, spanning the range from psychrophiles to hyperthermophiles. Several properties believed to be involved in the adaptation to different temperatures were calculated and compared for all ten structures. No sequence preferences, correlated with thermal stability, were apparent from the amino acid composition or from the analysis of the loops and secondary structure elements of the ten TIMs. A common feature for both psychrophilic and T. maritima TIM is the large number of salt bridges compared with the number found in mesophilic TIMs. In the two thermophilic TIMs, the highest amount of accessible hydrophobic surface is buried during the folding and assembly process.

A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism.

BACKGROUND: Thiolases are ubiquitous and form a large family of dimeric or tetrameric enzymes with a conserved, five-layered alphabetaalphabetaalpha catalytic domain. Thiolases can function either degradatively, in the beta-oxidation pathway of fatty acids, or biosynthetically. Biosynthetic thiolases catalyze the biological Claisen condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in a wide range of biosynthetic pathways, including those that generate cholesterol, steroid hormones, and various energy-storage molecules. RESULTS: The crystal structure of the tetrameric biosynthetic thiolase from Zoogloea ramigera has been determined at 2.0 A resolution. The structure contains a striking and novel 'cage-like' tetramerization motif, which allows for some hinge motion of the two tight dimers with respect to each other. The protein crystals were flash-frozen after a short soak with the enzyme's substrate, acetoacetyl-CoA. A reaction intermediate was thus trapped: the enzyme tetramer is acetylated at Cys89 and has a CoA molecule bound in each of its active-site pockets. CONCLUSIONS: The shape of the substrate-binding pocket reveals the basis for the short-chain substrate specificity of the enzyme. The active-site architecture, and in particular the position of the covalently attached acetyl group, allow a more detailed reaction mechanism to be proposed in which Cys378 is involved in both steps of the reaction. The structure also suggests an important role for the thioester oxygen atom of the acetylated enzyme in catalysis.

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power.

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.

Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1.

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.

The crystal structure of dienoyl-CoA isomerase at 1.5 A resolution reveals the importance of aspartate and glutamate sidechains for catalysis.

BACKGROUND: The degradation of unsaturated fatty acids is vital to all living organisms. Certain unsaturated fatty acids must be catabolized via a pathway auxiliary to the main beta-oxidation pathway. Dienoyl-coenzyme A (dienoyl-CoA) isomerase catalyzes one step of this auxiliary pathway, the isomerization of 3-trans,5-cis-dienoyl-CoA to 2-trans,4-trans-dienoyl-CoA, and is imported into both mitochondria and peroxisomes. Dienoyl-CoA isomerase belongs to a family of CoA-binding proteins that share the enoyl-CoA hydratase/isomerase sequence motif. RESULTS: The crystal structure of rat dienoyl-CoA isomerase has been determined at 1.5 A resolution. The fold closely resembles that of enoyl-CoA hydratase and 4-chlorobenzoyl-CoA dehalogenase. Dienoyl-CoA isomerase forms hexamers made up of two trimers. The structure contains a well ordered peroxisomal targeting signal type-1 which is mostly buried in the inter-trimer space. The active-site pocket is deeply buried and entirely hydrophobic, with the exception of the acidic residues Asp176, Glu196 and Asp204. Site-directed mutagenesis of Asp204 revealed that this residue is essential for catalysis. In a molecular modeling simulation, a molecule of 3-trans,5-cis-octadienoyl-CoA was docked into the active site. CONCLUSIONS: The structural data, supported by the mutagenesis data, suggest a reaction mechanism where Glu196 acts as a proton acceptor and Asp204 acts as a proton donor. Asp176 is paired with Glu196 and is important for optimizing the catalytic proton transfer properties of Glu196. In the predicted mode of substrate binding, an oxyanion hole stabilizes the transition state by binding the thioester oxygen. The presence of a buried peroxisomal targeting signal suggests that dienoyl-CoA isomerase is prevented from reaching its hexameric structure in the cytosol.

Insights into Src kinase functions: structural comparisons.

Recent structures of Src tyrosine kinases reveal complex mechanisms for regulation of enzymatic activity. The regulatory SH3 and SH2 domains bind to the back of the catalytic kinase domain via a linker region that joins the SH2 domain to the catalytic domain. Members of a subgroup of the Src kinase family show distinct features in this linker and in the loops that interact with it. Hydrophobicity of key residues in this region is important for intramolecular regulation. The kinases Abl, Btk and Csk seem to have the same molecular architecture as Src. Structural comparisons between serine/threonine and tyrosine kinases indicate a specific twisting mechanism involving the N- and C-terminal lobes of the catalytic domain. This motion could provide insights into the various mechanisms used to regulate kinase activity.

The crystal structure of enoyl-CoA hydratase complexed with octanoyl-CoA reveals the structural adaptations required for binding of a long chain fatty acid-CoA molecule.

The structure of the hexameric rat mitochondrial enoyl-Coenzyme A (CoA) hydratase, co-crystallised with the inhibitor octanoyl-CoA, has been refined at a resolution of 2.4 A. Enoyl-CoA hydratase catalyses the hydration of 2,3-unsaturated enoyl-CoA thioesters. In the crystal structure only four of the six active sites of the hexamer in the asymmetric unit are occupied with a ligand molecule, showing an unliganded and a liganded active site within the same crystal form. While the protein assembly and fold is identical to the previously solved acetoacetyl-CoA complex, differences are observed close to the fatty acid binding pocket due to the different nature of the ligands. The fatty acid tail of octanoyl-CoA is bound in an extended conformation. This is possible because a high B-factor loop, which separates in the acetoacetyl-CoA complex the binding pocket of the acetoacetyl-CoA fatty acid tail from the intertrimer space, has moved aside to allow binding of the longer octanoyl-CoA moiety. The movement of this loop opens a tunnel which traverses the complete subunit from the solvent space to the intertrimer space. The conformation of the catalytic residues is identical, in both structures as well as in the liganded and the unliganded active sites. In the unliganded active sites a water molecules is bound between the two catalytic glutamate, residues Glu144 and Glu164. After superposition of a liganded active site on an unliganded active site this water molecule is close to the carbon centre that becomes hydroxylated in the hydratase reaction. These findings support the idea that the active site is rigid and that the catalytic residues and the water molecule, as seen in the unliganded active site, are pre-positioned for very efficient catalysis.