Biomedical subjects

Y Lindqvist

Publications and source records attributed to Y Lindqvist.

At least 37 records · Page 2Linked to original sources

PubMed1999

Structure of dethiobiotin synthetase at 0.97 A resolution.

The crystal structure of the 224-residue protein dethiobiotin synthetase from Escherichia coli has been refined using X-ray diffraction data at 0.97 A resolution at 100 K. The model, consisting of 4143 protein atoms including 1859 H atoms and 436 solvent sites, was refined to a final R factor of 11.6% for all reflections, and has an estimated mean standard uncertainty for the atomic positions of 0.022 A, derived from inversion of the blocked matrix. The structure was refined with a full anisotropic model for the atomic displacement parameters using SHELX97. Stereochemical restraints were applied throughout the refinement. In the last cycles, the planarity of the peptide bonds was not restrained, resulting in a mean omega value of 179.6 degrees. Analysis of the most anisotropic regions of the protein shows that they form four clusters of residues. Alternate conformations for the side chains of 15 residues and for the main-chain atoms of six residues from three loops were included in the model. An analysis of C-HcO hydrogen bonds shows that such interactions occur rather frequently in DTBS; in total, 16 such hydrogen bonds were found. In the central beta-sheet, 13 C-HcO bonds between carbonyl O and Calpha H atoms were found. Other interactions of this type involve main-chain-side-chain and side-chain-side-chain C-HcO bonds. The model includes 436 water sites, of which 233 molecules form the first hydration shell. Analysis of the protein-solvent interactions shows that about one third of the accessible surface of the enzyme is not covered by ordered solvent. No difference in propensity for ordered solvent close to hydrophilic or hydrophobic surface areas was found. The comparison of the 100 K structure with the structure of the enzyme determined at room temperature shows several regions with different conformation, including areas in the active site, suggesting that structural transitions can occur during flash freezing. This observation questions one of the basic assumptions in the analysis of enzymatic reaction mechanisms using cryocrystallography.

Carbon-Nitrogen Ligases↗

PubMed1999

Tartrate-resistant bone acid phosphatase: large-scale production and purification of the recombinant enzyme, characterization, and crystallization.

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed in bone-resorbing osteoclasts and certain tissue macrophages in human tissues. The functions of TRAP in biological systems are not known. Elucidation of the three-dimensional structure of the active site could yield important information about the physiological substrate(s) of the enzyme. We have produced recombinant rat bone TRAP using a baculovirus expression vector system. The production was scaled up to a 30-l bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypeptide chain. Trypsin digestion resulted in a preparation where two subunits of approximately 23 kDa and approximately 16 kDa appeared after disulfide reduction. Trypsin digestion activated the enzyme. We generated monoclonal antibodies against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzyme could be separated by Mono-S cation-exchange chromatography. Crystals of TRAP have been obtained with ammonium sulfate/polyethylene glycol as precipitant. They belong to space group P212121 or P21212 with unit cell dimensions a = 57.2 A, b = 69.5 A, and c = 87.2 A and diffract to at least 2.2 A resolution. A packing density value of 2.55 A3/Da is consistent with one subunit in the asymmetric unit.

Acid Phosphatase↗

PubMed1998

Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 A resolution.

The three-dimensional structure of human tissue inhibitor of metalloproteinases-2 (TIMP-2) was determined by X-ray crystallography to 2.1 A resolution. The structure of the inhibitor consists of two domains. The N-terminal domain (residues 1-110) is folded into a beta-barrel, similar to the oligonucleotide/oligosaccharide binding fold otherwise found in certain DNA-binding proteins. The C-terminal domain (residues 111-194) contains a parallel stranded beta-hairpin plus a beta-loop-beta motif. Comparison of the structure of uncomplexed human TIMP-2 with that of bovine TIMP-2 bound to the catalytic domain of human MMP-14 suggests an internal rotation between the two domains of approximately 13 degrees upon binding to the protease. Furthermore, local conformational differences in the two structures that might be induced by formation of the protease-inhibitor complex have been found. The most prominent of these involves residues 27-40 of the A-B beta-hairpin loop. Structure-based alignment of amino acid sequences of representatives of the TIMP family maps the sequence differences mainly to loop regions, and some of these differences are proposed to be responsible for the particular properties of the various TIMP species.

Amino Acid Sequence↗

PubMed1998

Purification and preliminary X-ray crystallographic studies of recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli.

Recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli, a pyridoxal-phosphate-dependent aminotransferase, has been crystallized in space groups P21 and C2. Both crystal forms were obtained at pH 7.3 with 21% polyethylene glycol and 10% 2-propanol as precipitants. The cell dimensions were a = 130, b = 57.5, c = 117 A, beta = 110 degrees for the C2 crystals, and a = 58.4, b = 55.6, c = 121 A, beta = 96.9 degrees for the P21 crystals, which diffract to at least 2.6 and 2.0 A resolution, respectively.

Bacterial Proteins↗

PubMed1998

Crystallography and mutagenesis of transketolase: mechanistic implications for enzymatic thiamin catalysis.

The ThDP dependent enzyme transketolase is a convenient model system to study enzymatic thiamin catalysis. Crystallographic studies of the enzyme have identified the ThDP binding fold, the V-conformation of ThDP as the relevant conformation in enzymatic catalysis and details of enzyme-substrate interactions. Based on this structural information, the function of various active site residues in substrate binding and catalysis has been probed by site-directed mutagenesis.

Apoenzymes↗

PubMed1998

The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis.

BACKGROUND: The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy. The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols. RESULTS: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation. This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction. CONCLUSIONS: Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.

Amino Acid Sequence↗

PubMed1998

Snapshot of a phosphorylated substrate intermediate by kinetic crystallography.

The ATP-dependent enzyme dethiobiotin synthetase from Escherichia coli catalyses the formation of dethiobiotin from CO2 and 7, 8-diaminopelargonic acid. The reaction is initiated by the formation of a carbamate and proceeds through a phosphorylated intermediate, a mixed carbamic phosphoric anhydride. Here, we report the crystal structures at 1.9- and 1.6-A resolution, respectively, of the enzyme-MgATP-diaminopelargonic acid and enzyme-MgADP-carbamic-phosphoric acid anhydride complexes, observed by using kinetic crystallography. Reaction initiation by addition of either NaHCO3 or diaminopelargonic acid to crystals already containing cosubstrates resulted in the accumulation of the phosphorylated intermediate at the active site. The phosphoryl transfer step shows inversion of the configuration at the phosphorus atom, consistent with an in-line attack by the carbamate oxygen onto the phosphorus atom of ATP. A key feature in the structure of the complex of the enzyme with the reaction intermediate is two magnesium ions, bridging the phosphates at the cleavage site. These magnesium ions compensate the negative charges at both phosphate groups after phosphoryl transfer and contribute to the stabilization of the reaction intermediate.

Adenosine Triphosphate↗

PubMed1998

Crystal structure of beta-ketoacyl-acyl carrier protein synthase II from E.coli reveals the molecular architecture of condensing enzymes.

In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. The crystal structure of beta-ketoacyl synthase II from Escherichia coli has been determined with the multiple isomorphous replacement method and refined at 2.4 A resolution. The subunit consists of two mixed five-stranded beta-sheets surrounded by alpha-helices. The two sheets are packed against each other in such a way that the fold can be described as consisting of five layers, alpha-beta-alpha-beta-alpha. The enzyme is a homodimer, and the subunits are related by a crystallographic 2-fold axis. The two active sites are located near the dimer interface but are approximately 25 A apart. The proposed nucleophile in the reaction, Cys163, is located at the bottom of a mainly hydrophobic pocket which is also lined with several conserved polar residues. In spite of very low overall sequence homology, the structure of beta-ketoacyl synthase is similar to that of thiolase, an enzyme involved in the beta-oxidation pathway, indicating that both enzymes might have a common ancestor.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1998

Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis.

The crystal structures of two complexes of dethiobiotin synthetase, enzyme-diaminopelargonic acid-MgADP-AlF3 and enzyme-dethiobiotin-MgADP-Pi, respectively, have been determined to 1.8 A resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two-metal mechanism for this enzyme.

Adenosine Diphosphate↗

PubMed1998

The crystal structure of H-2Dd MHC class I complexed with the HIV-1-derived peptide P18-I10 at 2.4 A resolution: implications for T cell and NK cell recognition.

The structure of H-2Dd complexed with the HIV-derived peptide P18-I10 (RGPGRAFVTI) has been determined by X-ray crystallography at 2.4 A resolution. This MHC class I molecule has an unusual binding motif with four anchor residues in the peptide (G2, P3, R/K/H5, and I/L/F9 or 10). The cleft architecture of H-2Dd includes a deep narrow passage accomodating the N-terminal part of the peptide, explaining the obligatory G2P3 anchor motif. Toward the C-terminal half of the peptide, p5R to p8V form a type I' reverse turn; residues p6A to p9T, and in particular p7F, are readily exposed. The structure is discussed in relation to functional data available for T cell and natural killer cell recognition of the H-2Dd molecule.

Binding Sites↗

PubMed1997

Identification of catalytically important residues in yeast transketolase.

The possible roles of four histidine residues in the active site of yeast transketolase were examined by site-directed mutagenesis. Replacement of the invariant His69 with alanine yielded a mutant enzyme with 1.5% of the specific activity of the wild-type enzyme and with an increased KM for the donor. This residue is located at the bottom of the substrate cleft close to the C1 hydroxyl group of the donor substrate, and the side chain of His69 might be required for recognition of this hydroxyl group and possibly for maintenance of the proper orientation of the reaction intermediate, (alpha, beta-dihydroxyethyl)thiamin diphosphate. Amino acid replacements of His481 by alanine, serine, and glutamine resulted in mutant enzymes with significantly increased KM values for the donor substrate and specific activities of 4.4%, 1.9%, and 5.5% of the wild-type enzyme. The kinetic data suggest that this residue, although close to the C2 carbonyl oxygen of the substrate, is not absolutely required for stabilization of the negative charge that develops at this oxygen in the transition state. This points toward the 4'-NH2 group of the pyrimidine ring of thiamin diphosphate as the major source of charge stabilization. Mutations at positions His30 and His263 result in mutant enzymes severely impaired in catalytic activity (1.5% and less of the activity of wild-type transketolase). The KM value for the donor substrate was increased for the His30Ala mutant but remained unchanged in the His263Ala enzyme. The side chains of both residues interact with the C3 hydroxyl group of the donor substrate, and the results indicate that the two residues act in concert during proton abstraction of the C3 hydroxyl proton during catalysis.

Binding Sites↗

PubMed1997

Crystal structure of nitrile hydratase reveals a novel iron centre in a novel fold.

BACKGROUND: Nitrile hydratases are unusual metalloenzymes that catalyze the hydration of nitriles to their corresponding amides. They are used as biocatalysts in acrylamide production, one of the few commercial scale bioprocesses, as well as in environmental remediation for the removal of nitriles from waste streams. Nitrile hydratases are composed of two subunits, alpha and beta, and they contain one iron atom per alphabeta unit. We have determined the crystal structure of photoactivated iron-containing nitrile hydratase from Rhodococcus sp. R312 to 2.65 A resolution as a first step in the elucidation of its catalytic mechanism. RESULTS: The alpha subunit consists of a long N-terminal arm and a C-terminal domain that forms a novel fold. This fold can be described as a four layered structure, alpha-beta-beta-alpha, with unusual connectivities between the beta strands. The beta subunit also contains a long N-terminal extension, a helical domain, and a C-terminal domain that folds into a beta roll. The two subunits form a tight heterodimer that is the functional unit of the enzyme. The active site is located in a cavity at the subunit-subunit interface. The iron centre is formed by residues from the alpha subunit only-three cysteine thiolates and two mainchain amide nitrogen atoms are ligands. CONCLUSIONS: Nitrile hydratases contain a novel iron centre with a structure not previously observed in proteins; it resembles a hybrid of the iron centres of heme and Fe-S proteins. The low-spin electronic configuration presumably results in part from two Fe-amide nitrogen bonds. The structure is consistent with the metal ion having a role as a Lewis acid in the catalytic reaction.

Binding Sites↗

PubMed1997

Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position.

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Delta6-palmitoyl (16:0)-ACP desaturase with their equivalents from a Delta9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Delta6 or Delta9 positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Delta6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Delta9-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Delta9-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Delta9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.

Amino Acid Sequence↗

PubMed1997

Active site mutants of Escherichia coli dethiobiotin synthetase: effects of mutations on enzyme catalytic and structural properties.

Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41, implicated by the protein crystal structure studies of Escherichia coli DTBS, were mutated to determine their function in catalysis and substrate binding. Nine mutant enzymes, T11V, E12A, E12D, K15Q, K37L, K37Q, K37R, S41A, and S41C, were overproduced in an E. coli strain lacking a functional endogenous DTBS gene and purified to homogeneity. Replacement of Thr11 with valine resulted in a 24,000-fold increase in the Km(ATP) with little or no change in the Kd(ATP), KM(DAPA) and DTBS k(cat), suggesting an essential role for this residue in the steady-state affinity for ATP. The two Glu12 mutants showed essentially wild-type DTBS activity (slightly elevated k(cat)'s). Unlike wild-type DTBS, E12A had the same apparent KM(DAPA) at subsaturating and saturating ATP concentrations, indicating a possible role for Glu12 in the binding synergy between DAPA and ATP. The mutations in Lys15 and Lys37 resulted in loss of catalytic activity (0.01% and <0.9% of wild-type DTBS k(cat) for K15Q and the Lys37 mutant enzymes, respectively) and higher KM's for both DAPA (40-fold and >100-fold higher than wild-type for the K15Q and Lys37 mutant enzymes, respectively) and ATP (1800-fold and >10-fold higher than wild-type for K15Q and the K37 mutant enzymes, respectively). These results strongly suggest that Lys15 and Lys37 are crucial to both catalysis and substrate binding. S41A and S41C had essentially the same k(cat) as wild-type and had moderate increases in the DAPA and ATP KM and Kd (ATP) values. Replacement of Ser41 with cysteine resulted in larger effects than replacement with alanine. These data suggest that the H-bond between N7 of DAPA and the Ser41 side chain is not very important for catalysis. The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S41A, S41C, K37Q, and K37L, showed that the crystals were essentially isomorphous to that of the wild-type DTBS. The models of these mutant enzymes were well refined (1.9 -2.6 A) and showed good similarity to the wild-type enzyme (rmsd of C alpha atoms: 0.16-0.24 A). The crystal structure of S41C complexed with DAPA, Mn2+/Mg2+, and AMPPCP revealed a localized conformational change (rotations of side chains of Cys41 and Thr11) which can account for the changes in the kinetic parameters observed for S41C. The crystal structures of the Lys37 mutant enzymes showed that the positive charge of the side chain of Lys37 is indispensable. Mutations of Lys37 to either glutamine or leucine resulted in a shift of the metal ion (up to 0.5 A) together with side chains of other active site residues which could disrupt the subtle balance between the positive and negative charges in the active site. The conformational change of the phosphate binding loop (Gly8-X-X-X-X-X-Gly14-Lys15-Thr16) upon nucleotide binding observed previously [Huang, W., Jia, J., Gibson, K. J., Taylor, W. S., Rendina, A. R., Schneider, G., & Lindqvist, Y. (1995) Biochemistry 34, 10985] appears to be important to attain the proper active site scaffold.

Adenosine Triphosphate↗

PubMed1997

Trihydroxynaphthalene reductase from Magnaporthe grisea: realization of an active center inhibitor and elucidation of the kinetic mechanism.

Active trihydroxynaphthalene reductase (3HNR) is essential for the biosynthesis of fungal melanin by Magnaporthe grisea and is a focus of inhibitor design studies directed toward control of blast disease in rice. Tricyclazole, a preventative fungicide against rice blast, has been previously characterized as inhibiting 3HNR noncompetitively [Viviani, F., Vors, J. P., Gaudry, M., & Marquet, A. (1993) Bull. Soc. Chem. Fr. 136, 395-404] with respect to its naphthol substrate. Our steady-state kinetic and fluorescence titration studies show that instead the inhibitor binds competitively with respect to the naphthol substrate and that it binds to 3HNR forms with the preferences 3HNR.NADPH > 3HNR.NADP+ > 3HNR (unliganded): Kt = 15 nM, 0.56 microM, and Kd = 8.5 microM, respectively. Analysis of the frontier molecular orbitals of tricyclazole and NADP(H) provides a basis for the affinity differences of tricyclazole for 3HNR.NADP(H) enzyme forms. Fluorescence titrations show that NADPH and naphthol substrates form binary complexes with 3HNR [Kd(NADP+) = 38 microM and Kd(U7278, an alternate naphthol-like substrate) = 220 microM]. However, the overwhelmingly preferred order of productive binding is NADPH followed by naphthol substrate, as shown by the uncompetitive inhibition of 3HNR by tricyclazole with respect to NADPH. Consistent with this mechanism, the K(m)'s for the naphthol substrates U7278 and scytalone (5 and 6 microM, respectively) are much lower than the Kd's of the binary complexes. The partition ratio of U7278 and a physiological substrate (scytalone) was 95:1 and unchanged on varying 3HNR.NADP+/3HNR(unliganded), which is also consistent with the ordered mechanism. The pH dependence of the hydride transfer rate from U7278 to NADP+ was measured, as was the pH dependence of Kcat/K(m)(NADP+). Hydride transfer had a pH dependence which suggests a single deprotonated residue (pKa = 6.0) is required for catalysis. Khyd, the rate constant for hydride transfer, was 9-fold larger than Kcat with U7278 as a substrate. A burst in the pre-steady-state suggests that release of one or both of the products is rate limiting to Kcat at pH 7.0. The pH dependence of Kcat/K(m)(NADP+) indicates a requirement for a single deprotonated group and this ionization is assigned to the 2' phosphate of NADP+. 3HNR was found to be 800-fold more specific for NADP+ relative to NAD+. Analysis of sequence and structure [Anderson, A., Jordan, D. B., Schneider. G., & Lindqvist. Y. (1996) Structure 4, 1161-1170] reveals that 3HNR is a member of the short-chain dehydrogenase superfamily of enzymes.

Antifungal Agents↗

PubMed1997

Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis.

The three-dimensional structure of the quaternary complex of Saccharomyces cerevisiae transketolase, thiamin diphosphate, Ca2+, and the acceptor substrate erythrose-4-phosphate has been determined to 2.4 A resolution by protein crystallographic methods. Erythrose-4-phosphate was generated by enzymatic cleavage of fructose-6-phosphate. The overall structure of the enzyme in the quaternary complex is very similar to the structure of the holoenzyme; no large conformational changes upon substrate binding were found. The substrate binds in a deep cleft between the two subunits. The phosphate group of the substrate interacts with the side chains of the conserved residues Arg359, Arg528, His469, and Ser386 at the entrance of this cleft. The aldehyde moiety of the sugar phosphate is located in the vicinity of the C-2 carbon atom of the thiazolium ring of the cofactor. The aldehyde oxygen forms hydrogen bonds to the side chains of the residues His30 and His263. One of the hydroxyl groups of the sugar phosphate forms a hydrogen bond to the side chain of Asp477. The preference of the enzyme for donor substrates with D-threo configuration at the C-3 and C-4 positions and for alpha-hydroxylated acceptor substrates can be understood from the pattern of hydrogen bonds between enzyme and substrate. Amino acid replacements by site-directed mutagenesis of residues Arg359, Arg528, and His469 at the phosphate binding site yield mutant enzymes with considerable residual catalytic activity but increased Km values for the donor and in particular acceptor substrate, consistent with a role for these residues in phosphate binding. Replacement of Asp477 by alanine results in a mutant enzyme impaired in catalytic activity and with increased Km values for donor and acceptor substrates. These findings suggest a role for this amino acid in substrate binding and catalysis.

Circular Dichroism↗

PubMed1997

A flexible lid controls access to the active site in 1,3,8-trihydroxynaphthalene reductase.

The crystal structures of apo-1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea and a binary complex of the enzyme with NADPH have been determined to 2.8 A resolution. In both cases, the overall structure is preserved compared to the structure of the ternary complex of the enzyme with NADPH and an active site inhibitor. No electron density for the helix-loop-helix region comprising residues 214-244 is observed indicating structural disorder in this part of the apoenzyme and the binary complex. In the ternary complex, this region is in contact with NADPH and the inhibitor and closes off the active site. The observed increase in flexibility in the absence of the inhibitor indicates that this region acts as a lid which closes the active site upon binding of the inhibitor and, possibly the substrate, 1,3,8-trihydroxynaphthalene.

Ascomycota↗

PubMed1997

Crystal structure of the reduced Schiff-base intermediate complex of transaldolase B from Escherichia coli: mechanistic implications for class I aldolases.

Transaldolase catalyzes transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor. During catalysis, a Schiff-base intermediate between dihydroxyacetone and the epsilon-amino group of a lysine residue at the active site of the enzyme is formed. This Schiff-base intermediate has been trapped by reduction with potassium borohydride, and the crystal structure of this complex has been determined at 2.2 A resolution. The overall structures of the complex and the native enzyme are very similar; formation of the intermediate induces no large conformational changes. The dihydroxyacetone moiety is covalently linked to the side chain of Lys 132 at the active site of the enzyme. The Cl hydroxyl group of the dihydroxyacetone moiety forms hydrogen bonds to the side chains of residues Asn 154 and Ser 176. The C3 hydroxyl group interacts with the side chain of Asp 17 and Asn 35. Based on the crystal structure of this complex a reaction mechanism for transaldolase is proposed.

Catalysis↗