Aspects of the mechanism of catalysis of glucose oxidase: a docking, molecular mechanics and quantum chemical study.

The complex structure of glucose oxidase (GOX) with the substrate glucose was determined using a docking algorithm and subsequent molecular dynamics simulations. Semiempirical quantum chemical calculations were used to investigate the role of the enzyme and FAD co-enzyme in the catalytic oxidation of glucose. On the basis of a small active site model, substrate binding residues were determined and heats of formation were computed for the enzyme substrate complex and different potential products of the reductive half reaction. The influence of the protein environment on the active site model was estimated with a point charge model using a mixed QM/MM method. Solvent effects were estimated with a continuum model. Possible modes of action are presented in relation to experimental data and discussed with respect to related enzymes. The calculations indicate that the redox reaction of GOX differs from the corresponding reaction of free flavins as a consequence of the protein environment. One of the active site histidines is involved in substrate binding and stabilization of potential intermediates, whereas the second histidine is a proton acceptor. The former one, being conserved in a series of oxidoreductases, is also involved in the stabilization of a C4a-hydroperoxy dihydroflavin in the course of the oxidative half reaction.

Ta6Br(2+)12, a tool for phase determination of large biological assemblies by X-ray crystallography.

The title compound Ta6Br(2+)12 is of interest for the analysis of biological structures as a heavy-metal derivative with great potential for the structure determination of large protein systems. In macromolecular crystallography the phases of the measured structure factor amplitudes have to be determined. The most widely used method for novel structures is isomorphous replacement by introducing electron-rich compounds into the protein crystals. These compounds produce measurable changes of the diffraction intensities, which allow phase determination. We synthetized the Ta6Br(2+)12 cluster in high yields, crystallized it, and determined its crystal structure by X-ray diffraction analysis at atomic resolution. The cluster is a regular octahedron consisting of six metal atoms with 12 bridging bromine atoms along the 12 edges of the octahedron. The cluster is compact, of approximately spherical shape with about 4.3 A radius and highly symmetrical. One Ta6Br(2+)12 ion adds 856 electrons to a protein, a considerable contribution to the scattering power even of large proteins or multimeric systems. At low resolution all atoms of the cluster scatter in phase and act as a super heavy-atom, which is easy to locate in the difference Patterson map. We investigated its binding sites in the biologically significant high-resolution structures of an antibody V(L) domain, dimethyl sulfoxide reductase, GTP-cyclohydrolase I, and the proteasome. With the randomly oriented cluster, treated as a single site scatterer, phases could be used only up to 6 A resolution. In contrast, when the cluster is correctly oriented, phases calculated from its 18 atom sites can be used to high resolution. We present the atomic structure of the Ta6Br(2+)12, describe a method to determine its localization and orientation in the unit cell of protein crystals of two different proteins, and analyse its phasing power. We show that phases can be calculated to high resolution. The phase error is lower by more than 30 degrees compared to the single site approximation, using a resolution of 2.2 A. Furthermore, Ta6Br(2+)12 has two different strong anomalous scatterers tantalum and bromine to be used for phase determination.

The suitability of Ta6Br12(2+) for phasing in protein crystallography.

The title compound Ta6Br12(2+) is of interest for the analysis of biological structures as a heavy metal derivative with great potential for the structure determination of large protein systems. In macromolecular crystallography the phases of the measured structure factor amplitudes have to be determined by compounds that produce measurable changes of the diffraction intensities. Ta6Br12(2+) uniquely meets the demands of a heavy metal derivative and is an important tool for phase determination, especially considering the structure determination of large protein systems.

Feedback inhibition of dihydrodipicolinate synthase enzymes by L-lysine.

Dihydrodipicolinate synthase (DHDPS) is the first enzyme unique to the lysine biosynthetic pathway and is feedback regulated by L-lysine in plants and some bacteria. The allosteric binding site has been localized by X-ray crystallography and is in agreement with reported mutations of plant DHDPS enzymes, which confer insensitivity to feedback inhibition. Three possible elements of the mechanism of lysine inhibition are discussed.

Isolation, cloning, sequence analysis and X-ray structure of dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.

The periplasmic enzyme dimethyl sulfoxide/trimethylamine N-oxide reductase (DMSOR/TMAOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO). At a molybdenum redox centre, two single electrons are transferred from cytochrome c556 to the substrate, e.g. DMSO, generating dimethyl sulfide (DMS) and water. The operon encoding this enzyme was isolated, cloned and sequenced, and its chromosomal location determined. It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes. Degenerate primers were derived from short peptide sequences and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library. Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/TMAOR operon. By an optimized protein purification high yields (5 mg protein/l culture) with a specific activity of 30 U/mg were obtained. The molecular mass was experimentally determined by electrospray mass spectroscopy (MS) to be 85034 Da and from the deduced amino acid sequence of the apoenzyme to be 85033 Da. The enzyme was crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A diffracting beyond 1.8 A. The three-dimensional structure was solved by a combination of multiple isomorphous replacement (MIR) and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The sole bis(molybdopterin guanine dinucleotide)molybdenum cofactor (1541 Da) of the single chain protein has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide (MGD) molecule. In addition, two oxo ligands and the oxygen of a serine side chain are bound to the molybdenum ion.

Organization of the DMSO respiratory operon of Rhodobacter capsulatus and its consequences for homologous expression of DMSOR/TMAOR.

In this report we describe the organization of the respiratory operon from the non-sulfur photosynthetic bacterium Rhodobacter capsulatus and its consequences for homologous expression of recombinant dimethylsulfoxide/trimethylamine N-oxide reductase (DMSOR/TMAOR). This enzyme is of special interest since molybdopterin dinucleotide is its only cofactor. Overexpression of the dmsA gene and production of active enzyme is not possible in E. coli because this bacterium is unable to supply the required complex molybdopterin cofactor. To investigate the catalytic mechanism and binding of the cofactor by structure-based site-directed mutagenesis, and to ensure sufficient production and incorporation of the complex cofactor, we designed a system for homologous expression in R. capsulatus.

Structure and function of molybdopterin containing enzymes.

Molybdopterin containing enzymes are present in a wide range of living systems and have been known for several decades. However, only in the past two years have the first crystal structures been reported for this type of enzyme. This has represented a major breakthrough in this field. The enzymes share common structural features, but reveal different polypeptide folding topologies. In this review we give an account of the related spectroscopic information and the crystallographic results, with emphasis on structure-function studies.

Cloning, sequencing, crystallization and X-ray structure of glutathione S-transferase-III from Zea mays var. mutin: a leading enzyme in detoxification of maize herbicides.

Glutathione S-transferases (GSTs) are enzymes that inactivate toxic compounds by conjugation with glutathione and are involved in resistance towards drugs, antibiotics, insecticides and herbicides. Their ability to confer herbicide tolerance in plants provides a tool to control weeds in a wide variety of agronomic crops. GST-III was prepared from Zea mays var. mutin and its amino acid sequence was determined from two sets of peptides obtained by cleavage with endoprotease Asp-N and with trypsin, respectively. Recombinant GST-III was prepared by extraction of mRNA from plant tissue, transcription into cDNA, amplification by PCR and expression. It was crystallized and the crystal structure of the unligated form was determined at 2.2 A resolution. The enzyme forms a GST-typical dimer with one subunit consisting of 220 residues. Each subunit is formed of two distinct domains, an N-terminal domain consisting of a beta-sheet flanked by two helices, and a C-terminal domain, entirely helical. The dimeric molecule is globular with a large cleft between the two subunits. The amino acid sequence of GST-III and its cDNA sequence determined here show differences from sequences published earlier.

Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure.

DHDPS is the first enzyme unique to the lysine biosynthetic pathway in plants and bacteria and catalyses the formation of (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. It is feedback-regulated in plants by L-lysine. The crystal structure of Nicotiana sylvestris DHDPS with and without inhibitory lysine bound to the enzyme has been solved to a resolution of 2.8 A. The molecule is a homotetramer composed of a dimer of dimers. Comparison with the structure of Escherichia coli DHDPS showed a novel quaternary structure by a profound rearrangement of the dimers forming the tetramer. The crystal structure of the enzyme in the presence of L-lysine revealed substantial changes. These changes together with the novel quaternary structure provide a structural basis for the strong inhibition of plant DHDPS enzymes by L-lysine.

Isolation, cloning, sequence analysis and localization of the operon encoding dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.

The operon encoding the periplasmic enzymes dimethyl sulfoxide reductase (DMSOR) and trimethylamine N-oxide reductase (TMAOR) from the purple, non-sulfur, photosynthetic bacterium Rhodobacter capsulatus was isolated, cloned and sequenced, and its chromosomal location determined. It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes. Degenerate primers were derived from short peptide sequences generated by automated Edman degradation and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library. Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/ TMAOR operon. The promoter consisted of an A + T-rich region followed by a -35 region, a putative ribosome binding site, and a leader sequence of 13 amino acid residues. The transcription terminator was a G + C-rich dyad sequence capable of forming a hairpin structure, which may act rho-independently. An optimized protein purification of the wild-type enzyme is also described, giving high yields (5 mg protein per liter of culture) and a specific activity of 30 units/mg. The molecular mass was determined by electrospray mass spectrometry to be 85,034 Da; from the deduced amino acid sequence the molecular mass of the apoenzyme was 85,033 Da.

Crystal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1.88 A resolution.

The periplasmic dimethyl sulfoxide reductase (DMSOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide to dimethyl sulfide. At a molybdenum redox center, two single electrons are transferred from cytochrome C556 to the substrate dimethyl sulfoxide, generating dimethyl sulfide and (with two protons) water. The enzyme was purified and crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A. The crystals diffract beyond 1.8 A with synchrotron radiation. The three-dimensional structure was solved by a combination of multiple isomorphous replacement and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57,394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The bis(molybdopterin guanine dinucleotide) molybdenum cofactor (1541 Da) of the single chain protein (85,033 Da) has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide molecule. Three additional ligands, two oxo groups and the oxygen of a serine side-chain, are bound to the molybdenum ion. The second molybdopterin system is not part of the ligand sphere of the metal center with its sulfur atoms at distances of 3.5 A and 3.8 A away. It might be involved in electron shuttling from the protein surface to the molybdenum center.