The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

Analysis of newly detected mutations in the MCFD2 gene giving rise to combined deficiency of coagulation factors V and VIII.

Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive disorder characterized by mild-to-moderate bleeding and reduction in FV and FVIII levels in plasma. F5F8D is caused by mutations in one of two different genes, LMAN1 and MCFD2, which encode proteins that form a complex involved in the transport of FV and FVIII from the endoplasmic reticulum to the Golgi apparatus. Here, we report the identification of a novel mutation Asp89Asn in the MCFD2 gene in a Tunisian patient. In the encoded protein, this mutation causes substitution of a negatively charged aspartate, involved in several structurally important interactions, to an uncharged asparagine. To elucidate the structural effect of this mutation, we performed circular dichroism (CD) analysis of secondary structure and stability. In addition, CD analysis was performed on two missense mutations found in previously reported F5F8D patients. Our results show that all analysed mutant variants give rise to destabilized proteins and highlight the importance of a structurally intact and functional MCFD2 for the efficient secretion of coagulation factors V and VIII.

Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme.

Thioredoxin reductases (TrxRs) from mammalian cells contain an essential selenocysteine residue in the conserved C-terminal sequence Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme. Reduction by NADPH generates a selenolthiol, which is the active site in reduction of Trx. The three-dimensional structure of the SeCys498Cys mutant of rat TrxR in complex with NADP(+) has been determined to 3.0-A resolution by x-ray crystallography. The overall structure is similar to that of glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues directly interacting with the substrate glutathione disulfide in GR are conserved despite the failure of glutathione disulfide to act as a substrate for TrxR. The 16-residue C-terminal tail, which is unique to mammalian TrxR, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of TrxR with Trx suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. The C-terminal extension typical of mammalian TrxRs has two functions: (i) it extends the electron transport chain from the catalytic disulfide to the enzyme surface, where it can react with Trx, and (ii) it prevents the enzyme from acting as a GR by blocking the redox-active disulfide. Our results suggest that mammalian TrxR evolved from the GR scaffold rather than from its prokaryotic counterpart. This evolutionary switch renders cell growth dependent on selenium.

Structural basis for the thioredoxin-like activity profile of the glutaredoxin-like NrdH-redoxin from Escherichia coli.

NrdH-redoxin is a representative of a class of small redox proteins that contain a conserved CXXC motif and are characterized by a glutaredoxin-like amino acid sequence and thioredoxin-like activity profile. The crystal structure of recombinant Escherichia coli NrdH-redoxin in the oxidized state has been determined at 1.7 A resolution by multiwavelength anomalous diffraction. NrdH-redoxin belongs to the thioredoxin superfamily and is structurally most similar to E. coli glutaredoxin 3 and phage T4 glutaredoxin. The angle between the C-terminal helix alpha3 and strand beta4, which differs between thioredoxin and glutaredoxin, has an intermediate value in NrdH-redoxin. The orientation of this helix is to a large extent determined by an extended hydrogen-bond network involving the highly conserved sequence motif (61)WSGFRP(D/E)(67), which is unique to this subclass of the thioredoxin superfamily. Residues that bind glutathione in glutaredoxins are in general not conserved in NrdH-redoxin, and no glutathione-binding cleft is present. Instead, NrdH-redoxin contains a wide hydrophobic pocket at the surface, similar to thioredoxin. Modeling studies suggest that NrdH-redoxin can interact with E. coli thioredoxin reductase at this pocket and also via a loop that is complementary to a crevice in the reductase in a similar manner as observed in the E. coli thioredoxin-thioredoxin reductase complex.

Crystallization and preliminary X-ray crystallographic studies of recombinant thermoresistant gluconate kinase GntK from Escherichia coli.

The thermoresistant gluconate kinase GntK from Escherichia coli, an essential enzyme in gluconate metabolism, has been expressed, purified and crystallized. For crystallization, the hanging-drop vapour-diffusion method was used with polyethylene glycol (PEG) 6000 and lithium chloride as precipitants. Three crystal forms belonging to the monoclinic space group C2 or the orthorhombic space groups P2(1)2(1)2(1) and P2(1)2(1)2 were obtained. The unit-cell parameters are a = 75.0, b = 79.3, c = 70.2 A, beta = 105.3 degrees (C2), a = 52.0, b = 79.3, c = 89.8 A (P2(1)2(1)2(1)) and a = 70.1, b = 74.1, c = 78.9 A (P2(1)2(1)2). In all three crystal forms, there are two molecules in the asymmetric unit; the different forms occur in the same crystallization drop. The crystals diffract to at least 2.0 A using synchrotron radiation.

Structural enzymology of biotin biosynthesis.

Over the last years, significant progress has been made in the understanding of the genetics and enzymology of the biosynthetic pathway of the vitamin biotin. The enzymes catalyzing the last four steps of this pathway, from pimeloyl-CoA to biotin, provide an ensemble of intriguing reaction mechanisms, which are presently being unravelled. The three-dimensional structures for three of these enzymes are known and provide a framework to which on-going mechanistic studies can be related.

Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil.

Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5-fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2x 111 kDa) determined at 1.9 A resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe-4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe-S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5-fluorouracil reveals the architecture of the substrate-binding sites and residues responsible for recognition and binding of the drug.

Crystallization and preliminary X-ray study of pig liver dihydropyrimidine dehydrogenase.

Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting reaction in pyrimidine catabolism. The enzyme contains one FMN, one FAD and four Fe-S clusters per subunit of 1025 amino acids as prosthetic groups. It is also the major determinant of bioavailability and toxicity of 5-fluorouracil, a chemotherapeutic agent widely used in the treatment of solid tumors. Crystals of this enzyme diffracting to at least 2.5 A have been obtained by the hanging-drop vapour-diffusion method and belong to space group P2(1) (unit-cell parameters a = 82.0, b = 159.3, c = 163.6 A, beta = 96.1 degrees ), with two homodimers per asymmetric unit.

The crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Synechocystis sp. at 1.54 A resolution and its relationship to other condensing enzymes.

Condensing enzymes, catalyzing the formation of carbon-carbon bonds in several biosynthetic pathways, have lately been recognized as potential drug targets against cancer and tuberculosis, as crucial for combinatorial biosynthesis of antibiotics and related compounds, and as determinants of plant oil composition. beta-Ketoacyl-ACP synthases (KAS) are the condensing enzymes present in the fatty acid biosynthesis pathway and are able to elongate an acyl chain bound to either co-enzyme A (CoA) or acyl carrier protein (ACP) with a two-carbon unit derived from malonyl-ACP. Several isoforms of KAS with different substrate specificity are present in most species. We have determined the crystal structure of KAS II from Synechocystis sp. PCC 6803 to 1.54 A resolution giving a detailed description of the active site geometry. In order to analyze the structure-function relationships in this class of enzymes in more detail, we have compared all presently known three-dimensional structures of condensing enzymes from different pathways. The comparison reveals that these enzymes can be divided into three structural and functional classes. This classification can be related to variations in the catalytic mechanism and the set of residues in the catalytic site, e.g. due to differences in the nature of the second substrate providing the two-carbon elongation unit. The variation in the acyl-carrier (ACP or CoA) specificity might also be connected to this classification and residues involved in ACP binding in structure class 2 can be suggested based on the comparison. Finally, the two subunits in the dimer contribute differently to formation of the substrate binding-pocket in the three structural classes.

Crystal structure of saccharopine reductase from Magnaporthe grisea, an enzyme of the alpha-aminoadipate pathway of lysine biosynthesis.

BACKGROUND: The biosynthesis of the essential amino acid lysine in higher fungi and cyanobacteria occurs via the alpha-aminoadipate pathway, which is completely different from the lysine biosynthetic pathway found in plants and bacteria. The penultimate reaction in the alpha-aminoadipate pathway is catalysed by NADPH-dependent saccharopine reductase. We set out to determine the structure of this enzyme as a first step in exploring the structural biology of fungal lysine biosynthesis. RESULTS: We have determined the three-dimensional structure of saccharopine reductase from the plant pathogen Magnaporthe grisea in its apo form to 2.0 A resolution and as a ternary complex with NADPH and saccharopine to 2.1 A resolution. Saccharopine reductase is a homodimer, and each subunit consists of three domains, which are not consecutive in amino acid sequence. Domain I contains a variant of the Rossmann fold that binds NADPH. Domain II folds into a mixed seven-stranded beta sheet flanked by alpha helices and is involved in substrate binding and dimer formation. Domain III is all-helical. The structure analysis of the ternary complex reveals a large movement of domain III upon ligand binding. The active site is positioned in a cleft between the NADPH-binding domain and the second alpha/beta domain. Saccharopine is tightly bound to the enzyme via a number of hydrogen bonds to invariant amino acid residues. CONCLUSIONS: On the basis of the structure of the ternary complex of saccharopine reductase, an enzymatic mechanism is proposed that includes the formation of a Schiff base as a key intermediate. Despite the lack of overall sequence homology, the fold of saccharopine reductase is similar to that observed in some enzymes of the diaminopimelate pathway of lysine biosynthesis in bacteria. These structural similarities suggest an evolutionary relationship between two different major families of amino acid biosynthetic pathway, the glutamate and aspartate families.

Cloning, expression, purification and crystallization of saccharopine reductase from Magnaporthe grisea.

The gene coding for saccharopine reductase (E.C. 1.5.1.10), an enzyme of the alpha-aminoadipic pathway of lysine biosynthesis in the pathogenic fungus Magnaporthe grisea, was cloned and expressed in Escherichia coli. The purified enzyme was crystallized in space groups C2 and C222(1) using ammonium sulfate pH 4.8 or PEG 6000 pH 4. 1 as precipitants. The unit-cell parameters are a = 115.0, b = 56.6, c = 74.3 A, beta = 111.1 degrees for space group C2, and a = 89.3, b = 119.0, c = 195.9 A for space group C222(1). The crystals diffract to resolutions of 2.0 A (C2) and 2.4 A (C222(1)) at synchrotron sources.

The manifold of vitamin B6 dependent enzymes.

Pyridoxal-5'-phosphate (vitamin B6) binding enzymes form a large superfamily that contains at least five different folds. The availability of an increasing number of known three-dimensional structures for members of this superfamily has allowed a detailed structural classification. Most progress has been made with the fold type I or aspartate aminotransferase family.

Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.

beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.

Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5'-phosphate-dependent enzymes.

The three-dimensional structure of diaminopelargonic acid synthase, a vitamin B6-dependent enzyme in the pathway of the biosynthesis of biotin, has been determined to 1.8 A resolution by X-ray crystallography. The structure was solved by multi-wavelength anomalous diffraction techniques using a crystal derivatized with mercury ions. The protein model has been refined to a crystallographic R -value of 17.5% (R -free 22.6%). Each enzyme subunit consists of two domains, a large domain (residues 50-329) containing a seven-stranded predominantly parallel beta-sheet, surrounded by alpha-helices, and a small domain comprising residues 1-49 and 330-429. Two subunits, related by a non-crystallographic dyad in the crystals, form the homodimeric molecule, which contains two equal active sites. Pyridoxal-5'-phosphate is bound in a cleft formed by both domains of one subunit and the large domain of the second subunit. The cofactor is anchored to the enzyme by a covalent linkage to the side-chain of the invariant residue Lys274. The phosphate group interacts with main-chain nitrogen atoms and the side-chain of Ser113, located at the N terminus of an alpha-helix. The pyridine nitrogen forms a hydrogen bond to the side-chain of the invariant residue Asp245. Electron density corresponding to a metal ion, most likely Na(+), was found in a tight turn at the surface of the enzyme. Structure analysis reveals that diaminopelargonic acid synthase belongs to the family of vitamin B6-dependent aminotransferases with the same fold as originally observed in aspartate aminotransferase. A multiple structure alignment of enzymes in this family indicated that they form at least six different subclasses. Striking differences in the fold of the N-terminal part of the polypeptide chain are one of the hallmarks of these subclasses. Diaminopelargonic acid synthase is a member of the aminotransferase subclass III. From the structure of the non-productive complex of the holoenzyme with the substrate 7-keto-8-aminopelargonic acid the location of the active site and residues involved in substrate binding have been identified.

Three-dimensional structure of a mammalian purple acid phosphatase at 2.2 A resolution with a mu-(hydr)oxo bridged di-iron center.

The crystal structure of purple acid phosphatase from rat bone has been determined by molecular replacement and the structure has been refined to 2.2 A resolution to an R -factor of 21.3 % (R -free 26.5 %). The core of the enzyme consists of two seven-stranded mixed beta-sheets, with each sheet flanked by solvent-exposed alpha-helices on one side. The two sheets pack towards each other forming a beta-sandwich. The di-iron center, located at the bottom of the active-site pocket at one edge of the beta-sandwich, contains a mu-hydroxo or mu-oxo bridge and both metal ions are observed in an almost perfect octahedral coordination geometry. The electron density map indicates that a mu-(hydr)oxo bridge is found in the metal center and that at least one solvent molecule is located in the first coordination sphere of one of the metal ions. The crystallographic study of rat purple acid phosphatase reveals that the mammalian enzymes are very similar in overall structure to the plant enzymes in spite of only 18 % overall sequence identity. In particular, coordination and geometry of the iron cluster is preserved in both enzymes and comparison of the active-sites suggests a common mechanism for the mammalian and plant enzymes. However, significant differences are found in the architecture of the substrate binding pocket.

Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed.

Matrix metalloproteinases (MMPs) catalyze extracellular matrix degradation. Control of their activity is a promising target for therapy of diseases characterized by abnormal connective tissue turnover. MMPs are expressed as latent proenzymes that are activated by proteolytic cleavage that triggers a conformational change in the propeptide (cysteine switch). The structure of proMMP-2 reveals how the propeptide shields the catalytic cleft and that the cysteine switch may operate through cleavage of loops essential for propeptide stability.

High-resolution structures of scytalone dehydratase-inhibitor complexes crystallized at physiological pH.

Scytalone dehydratase is a molecular target of inhibitor design efforts aimed at preventing the fungal disease caused by Magnaporthe grisea. A method for cocrystallization of enzyme with inhibitors at neutral pH has produced several crystal structures of enzyme-inhibitor complexes at resolutions ranging from 1.5 to 2.2 A. Four high resolution structures of different enzyme-inhibitor complexes are described. In contrast to the original X-ray structure of the enzyme, the four new structures have well-defined electron density for the loop region comprising residues 115-119 and a different conformation between residues 154 and 160. The structure of the enzyme complex with an aminoquinazoline inhibitor showed that the inhibitor is in a position to form a hydrogen bond with the amide of the Asn131 side chain and with two water molecules in a fashion similar to the salicylamide inhibitor in the original structure, thus confirming design principles. The aminoquinazoline structure also allows for a more confident assignment of donors and acceptors in the hydrogen bonding network. The structures of the enzyme complexes with two dichlorocyclopropane carboxamide inhibitors showed the two chlorine atoms nearly in plane with the amide side chain of Asn131. The positions of Phe53 and Phe158 are significantly altered in the new structures in comparison to the two structures obtained from crystals grown at acidic pH. The multiple structures help define the mobility of active site amino acids critical for catalysis and inhibitor binding.

Murine class I major histocompatibility complex H-2Dd: expression, refolding and crystallization.

A truncated soluble form of the murine class I major histocompatibility antigen complex H-2Dd was cloned using an Escherichia coli based system. It was expressed, refolded in vitro and crystallized in a complex with murine beta2 microglobulin and the peptide RGPGRAFVTI from the V3-loop of the gp160 HIV-1 protein. Crystals belonging to the space group P212121 with cell dimensions a = 51.3, b = 92.5, c = 108.8 A were obtained using two different crystallization conditions. The crystals contain one complex per asymmetric unit and diffract to at least 2.4 A resolution.

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.