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We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the − 3/+ 3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the β-elimination on [-,2)-α- l -rhamno-(1,4)-α- d -galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis.

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be characterised and provides evidence for bifunctionality of dCTP deaminase occurring outside the Archaea kingdom. A steady-state kinetic analysis revealed that the affinity for dCTP and deoxyuridine triphosphate as substrates for the synthesis of deoxyuridine monophosphate were very similar, a result that contrasts that obtained previously for the archaean Methanocaldococcus jannaschii enzyme, which showed approximately 10-fold lower affinity for deoxyuridine triphosphate than for dCTP. The crystal structures of the enzyme in complex with the inhibitor, thymidine triphosphate, and the apo form have been solved. Comparison of the two shows that upon binding of thymidine triphosphate, the disordered C-terminal arranges as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site. In the inactive conformation dephosphorylation cannot take place due to the absence of a water molecule otherwise hydrogen-bonded to O2 of the α-phosphate.

The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein CI, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P(L) and the lysogenic promoter P(R) by binding to multiple operator sites on the DNA. In this study, we used a small bistable genetic switch element from phage TP901-1 to study the effect of cI deletions in vivo and showed that 43 amino acids could be removed from the C-terminal end of CI without destroying the ability of CI to repress transcription from the P(L) or the bistable switch properties. We showed that a helix-turn-helix motif located in the N-terminal part of CI is involved in DNA binding by introducing specific point mutations. Purification of CI and truncated forms of CI followed by analytical gel filtration and chemical cross-linking demonstrated that the C-terminal end of CI was required for oligomerization and that CI may exist as a hexamer in solution. Furthermore, expression and purification of the C-terminal part of CI (amino acids 92-180) showed that this part of the protein contained all the amino acids required to form an oligomer with an apparent molecular weight corresponding to a hexamer. We found that the C-terminal end of CI was required for de-repression of the P(L) following SOS induction, suggesting that the hexameric form of CI is needed for this or that this part of the protein is involved in the interaction with host proteins. By using small-angle X-ray scattering, we show for the first time the overall solution structure of a full-length wild-type bacteriophage repressor at low resolution revealing that the TP901-1 repressor forms a flat oligomer, most probably a trimer of dimers.

The syntheses of the square-planar platinum(II) complexes [Pt(L)(tba)] (L = 5,5′-dichloro-2,2′-dihydroxyazobenzenate (dhab), (5-chloro-2-hydroxyphenylazo)-3-oxo-N-phenylbutanamidate (hpab) or (5-chloro-2-hydroxyphenylazo)-2-naphtholate (hpan) and (tba) = tributylamine) is reported, together with those of the complete set of analogous complexes [M(L)(py)] (M = nickel(II), palladium(II), platinum(II) and (py) = pyridine). The coordinating nitrogen has been assigned to the azo-nitrogen attached to the 5-chloro-2-hydroxyphenyl substituent in both [Ni(hpab)(py)] and [Pt(hpan)(tba)] by single crystal X-ray diffraction methods. It has been established that complexes with different group 10 metals are iso-structural by isomorphology of the crystals as determined by X-ray powder diffraction studies on the complexes containing pyridine in the fourth coordination site. Furthermore, we present a method to determine the coordination geometry by comparison of calculated 13C chemical shifts for possible coordination modes optimized by ab initio methods with experimentally measured 13C chemical shifts.

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg115 and Glu138, were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg2+, and the mutant enzyme also with the substrate dCTP and Mg2+. The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg2+ that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.

The flavoenzymes dihydroorotate dehydrogenases (DHODs) catalyze the fourth and only redox step in the de novo biosynthesis of UMP. Enzymes belonging to class 2, according to their amino acid sequence, are characterized by having a serine residue as the catalytic base and a longer N terminus. The structure of class 2 E. coli DHOD, determined by MAD phasing, showed that the N-terminal extension forms a separate domain. The catalytic serine residue has an environment differing from the equivalent cysteine in class 1 DHODs. Significant differences between the two classes of DHODs were identified by comparison of the E. coli DHOD with the other known DHOD structures, and differences with the class 2 human DHOD explain the variation in their inhibitors.

Background: β-ketoacyl-acyl carrier protein synthase (KAS) I is vital for the construction of the unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids. The new carbon-carbon bonds are created by KAS I in a Claisen condensation performed in a three-step enzymatic reaction. KAS I belongs to the thiolase fold enzymes, of which structures are known for five other enzymes. Results: Structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates have been determined to 2.40 and 1.85 A resolution, respectively. The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer. A detailed model is proposed for the catalysis of KAS I. Of the two histidines required for decarboxylation, one donates a hydrogen bond to the malonyl thioester oxo group, and the other abstracts a proton from the leaving group. Conclusions: The same mechanism is proposed for KAS II, which also has a Cys-His-His active site triad. Comparison to the active site architectures of other thiolase fold enzymes carrying out a decarboxylation step suggests that chalcone synthase and KAS III with Cys-His-Asn triads use another mechanism in which both the histidine and the asparagine interact with the thioester oxo group. The acyl binding pockets of KAS I and KAS II are so similar that they alone cannot provide the basis for their differences in substrate specificity.

Background: The fourth step and only redox reaction in pyrimidine de novo biosynthesis is catalyzed by the flavoprotein dihydroorotate dehydrogenase (DHOD). Based on their sequences, DHODs are grouped into two major families. Lactococcus lactis is one of the few organisms with two DHODs, A and B, belonging to each of the two subgroups of family 1. The B enzyme (DHODB) is a prototype for DHODs in Gram-positive bacteria that use NAD + as the second substrate. DHODB is a heterotetramer composed of two different proteins (PyrDB and PyrK) and three different cofactors: FMN, FAD, and a [2Fe-2S] cluster. Results: Crystal structures have been determined for DHODB and its product complex. The DHODB heterotetramer is composed of two closely interacting PyrDB-PyrK dimers with the [2Fe-2S] cluster in their interface centered between the FMN and FAD groups. Conformational changes are observed between the complexed and uncomplexed state of the enzyme for the loop carrying the catalytic cysteine residue and one of the lysines interacting with FMN, which is important for substrate binding. Conclusions: A dimer of two PyrDB subunits resembling the family 1A enzymes forms the central core of DHODB. PyrK belongs to the NADPH ferredoxin reductase superfamily. The binding site for NAD + has been deduced from the similarity to these proteins. The orotate binding in DHODB is similar to that in the family 1A enzymes. The close proximity of the three redox centers makes it possible to propose a possible electron transfer pathway involving residues conserved among the family 1B DHODs.

A steady state kinetic investigation of the P(i) activation of 5-phospho-d-ribosyl alpha-1-diphosphate synthase from Escherichia coli suggests that P(i) can bind randomly to the enzyme either before or after an ordered addition of free Mg(2+) and substrates. Unsaturation with ribose 5-phosphate increased the apparent cooperativity of P(i) activation. At unsaturating P(i) concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with P(i) directs the subsequent ordered binding of Mg(2+) and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg(2+) and substrates via a slow pathway where P(i) binds to the enzyme last. The random mechanism for P(i) binding was further supported by studies with competitive inhibitors of Mg(2+), MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying P(i) at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing P(i) concentrations. Results from ADP inhibition of P(i) activation suggest that these effectors compete for binding to a common regulatory site.

The crystal structures of racemic and optically active cis-3-hydroxy-2-(4-methoxyphenyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (C16H15NO3S) were determined to investigate the enantiomer associations formed in the solid state. X-ray diffraction data were collected with Cu Kα ( λ = 1.54184 A ) radiation at 122.0(5) K. Optically active: monoclinic, space group P21, with a = 7.8605(8), b = 16.148(2), c = 11.3477(11) A , β = 97.607(8)°, V = 1427.7(3) A 3 , Z = 4, Dx = 1.402 g cm−3, μ = 20.53 cm−1 refined to R = 0.0282 for 2922 observed reflections. Racemic: monoclinic, space group P 2 1 n , with a = 10.3322(14), b = 7.7110(11), c = 18.901(3) A , β = 99.781(13)°, V = 1484.0(4) A 3 , Z = 4, Dx = 1.349 g cm−3, μ = 19.75 cm−1, refined to R = 0.0277 for 2909 observed reflections. In both structures the strongest intermolecular interactions are NH⋯O and OH⋯O hydrogen bonds formed between molecules of identical chirality. The racemate and enantiomer crystal structures differ in that there are two different conformers in the crystal structure of the optically pure compound, in the symmetry relation between molecules connected by strong hydrogen bonds, and in the formation of weak CH⋯O hydrogen bonds. The structure determinations do not support the formation of the hydrogen-bonded dimers that had been proposed to cause the difference in NMR chemical shifts of the methoxy protons.

The copper(II), nickel(II) and cobalt(II) complexes of new S 2 N 2 ligands on Schiff bases of 4-benzoyl-3-methyl-1-phenyl-2-pyrazoline-5-thione and various diamines were prepared and characterized. Using different diamines it is possible to control the geometry around the central metal atom. An X-ray structure determination of a Cu(II) S 2 N 2 complex showed the (NCuS, NCuS) dihedral angle to be 52.11° and the following distances: CuS1 2.2274, CuS2 2.2089, CuN13 1.973, CuN23 1.998 A.

Solid-state 13 C and 2 H NMR spectra are found to very suitable for characterizing the short O H...O hydrogen bonds observed in acid salts of dicarboxylic acids. The majority of the investigated compounds are acid salts of malonic, succinic and tartaric acid with monovalent cations derived from alkali metals and small aliphatic amines. They include systems with symmetric and asymmetric hydrogen bonds. Accurate structural information about their geometry is available from low-temperature X-ray diffraction data. The 13 C chemical shifts of the C atoms in the different carboxy groups display a linear variation with the absolute difference between the two C O bond lengths. Theoretical ab initio calculations for model systems showed that the nuclear quadrupole coupling constant NQCC for 2 H increases with increasing asymmetry of the hydrogen-bonded system. NQCC values for 2 H in the short symmetric hydrogen-bonded systems are in the range 53–59 kHz compared with the larger values of up to 166kHz found in systems with longer asymmetric hydrogen bonds. The 2 H NQCC values display a perfect linear dependence on the asymmetry of the hydrogen bond. 2 H NQCC decreases with decreasing temperature in the symmetric hydrogen bonds showing that the corresponding potential has a single well.

Four circle neutron diffraction data were recorded for a single crystal of KMnCl3 cooled to 110 K and 10 K, respectively. Analysis of the data measured at 110 K showed that they correspond to the diffraction from a twinned crystal of α-KMnCl3. The twinning explains the luminescence behaviour of the crystal. At 110 K the cell dimensions corresponding to the orthorhombic space group Pnma are a = 7.05(3) A , b = 9.90(2) A and c = 6.96(1) A . The two grains are oriented with the a-axis from one grain parallel to the c-axis from the other. The relative proportions of the two grains were included in the refinement based on 170 observed reflections to R -values: R = 0.116, Rw = 0.085. The second data set was measured well below the transition temperature (TN = 98 K) where α-KMnCl3 transforms into a simple antiferromagnetic structure. An analysis of the magnetic intensities showed that the magnetic moment is parallel to the crystallographic c-axis and has the value (4.0 ± 0.1)μB in agreement with previous powder work.

Crystals suitable for an X-ray diffraction investigation have been obtained of recombinant Coprinus cinereus peroxidase expressed in Aspergillus oryzae . The crystals were grown by the hanging drop method with polyethylene glycol 6000 as the precipitant. A pH range from 6·2 to 8·0 and CaCl 2 or MgCl 2 present at a concentration of 0·35 M were essential for the crystal growth. A metastable monoclinic modification can be obtained under certain conditions, and with variations in temperature they are transformed into a stable orthorhombic modification. With CaCl 2 as the additive, the unit cell dimensions were a = 74·9 A, b = 76·8 A and c = 128·2 A. With two peroxidase molecules per asymmetric unit, the solvent content is 49% (v/v). In the diffraction pattern, the reflections Okl are systematically very weak for k = 2 n + 1. Combined with an analysis of the Patterson function, this showed that the two independent molecules are related by the pseudo-translational symmetry 0·29a+0·5b. The possible space groups are P 2 1 2 1 2 1 or P 2 1 22 1 because of this pseudosymmetry. The crystals diffract to a resolution of 2·9 A.

Recent studies have shown 2-phenylazopyridine (Azpy) to be a useful departure from polypyridyl type ligands in coordination chemistry; frequently lower oxidation states are stabilized by this ligand. We have prepared and studied [Cr(Azpy)2Cl2] which is surprisingly inert for a chromium(II) complex. The compound crystallizes in the space group P21/c with a = 7.953(3), b = 21.189(11), c = 12.922(12) A, β = 114.80(5)° and Z = 4. Based on 6777 reflections the structure has been refined to R = 0.030 and Rw = 0.038. The complex is six-coordinated with cis-chlorides, cis-azo and trans-pyridyl groups. The overall symmetry is close to C2. The azo NN distances are elongated to 1.282 and 1.314 A, significantly longer than observed in free azo groups. The magnetic moment of 2.8 BM is independent of temperature indicating an orbitally non-degenerate low-spin ground state. In acetonitrile the spectrum shows a band in the red at 1470 nm which is assigned as ligand-metal charge transfer, consistent with earlier interpretations on similar compounds. The compound is stable to air oxidation in acetonitrile or methylene chloride; cyclic voltammetry in these solvents yields potentials of 0.508 and −0.185 V, both appearing to be one electron transfers. The complex is a non-electrolyte in acetonitrile, nearly unchanged over 24 h. However, chloride ions are slowly replaced at 50 °C in this solvent by 2,2′-bipyridyl; this reaction is pseudo-first order in complex with a rate constant of 4.0 ± 0.3 × 10−7 s−1. All data indicate the presence of a strong chromium-Azpy pi-interaction.