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The core of β-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. β-Lactam-producing bacteria, includingStreptomyces clavuligerus, synthesize the precursor of the amino acid α-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form α-aminoadipate semialdehyde) to α-aminoadipic acid, is catalysed by the NAD+-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH fromS. clavuligerusin its apo form and in complexes with the cofactor NAD+, the product α-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD+binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.

A correlation between the conformation of the gating residues and the size and shape of the bound compound suggests a metabolite-driven mechanism for transport across the carboxysome shell., Cyanobacterial CO2 fixation is promoted by encapsulating and co-localizing the CO2-fixing enzymes within a protein shell, the carboxysome. A key feature of the carboxysome is its ability to control selectively the flux of metabolites in and out of the shell. The β-carboxysome shell protein CcmP has been shown to form a double layer of pseudohexamers with a relatively large central pore (~13 Å diameter), which may allow passage of larger metabolites such as the substrate for CO2 fixation, ribulose 1,5-bisphosphate, through the shell. Here we describe two crystal structures, at 1.45 Å and 1.65 Å resolution, of CcmP from Synechococcus elongatus PCC7942 (SeCcmP). The central pore of CcmP is open or closed at its ends, depending on the conformation of two conserved residues, Glu69 and Arg70. The presence of glycerol resulted in a pore that is open at one end and closed at the opposite end. When glycerol was omitted, both ends of the barrel became closed. A binding pocket at the interior of the barrel featured residual density with distinct differences in size and shape depending on the conformation, open or closed, of the central pore of SeCcmP, suggestive of a metabolite-driven mechanism for the gating of the pore.

The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 A resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB.

Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 A resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.

Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V(c) (V(max) for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the K(m) for CO(2) and O(2) was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 degrees C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 degrees C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of beta-strand 1 of the alpha/beta-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel of ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in discriminating between CO2 and O2. Genetic screening in Chlamydomonas reinhardtii previously identified a loop-6 V331A substitution that decreases carboxylation and CO2/O2 specificity. Revertant selection identified T342I and G344S substitutions that restore photosynthetic growth by increasing carboxylation and specificity of the V331A enzyme. In numerous X-ray crystal structures, loop 6 is closed or open depending on the activation state of the enzyme and the presence or absence of ligands. The carboxy terminus folds over loop 6 in the closed state. To study the molecular basis for catalysis, directed mutagenesis and chloroplast transformation were used to create T342I and G344S substitutions alone. X-ray crystal structures were then solved for the V331A, V331A/T342I, T342I, and V331A/G344S enzymes, as well as for a D473E enzyme created to assess the role of the carboxy terminus in loop-6 closure. V331A disturbs a hydrophobic pocket, abolishing several van der Waals interactions. These changes are complemented by T342I and G344S, both of which alone cause decreases in CO2/O2 specificity. In the V331A/T342I revertant enzyme, Arg339 main-chain atoms are displaced. In V331A/G344S, alpha-helix 6 is shifted. D473E causes disorder of the carboxy terminus, but loop 6 remains closed. Interactions between a transition-state analogue and several residues are altered in the mutant enzymes. However, active-site Lys334 at the apex of loop 6 has a normal conformation. A variety of subtle interactions must be responsible for catalytic efficiency and CO2/O2 specificity.

Central steps in the biosynthetic pathways of some of the most commonly used antibiotics, the cephalosporins, are catalysed by 2-oxoglutarate (2OG)-dependent oxygenases. Deacetoxycephalosporin C synthase (DAOCS) catalyses the 2OG-dependent oxidative expansion of the five-membered thiazolidine ring of the penicillin nucleus into the six-membered dihydrothiazine ring of the cephalosporin nucleus. DAOCS uses dioxygen to create a reactive iron–oxygen intermediate from ferrous ion to drive the reaction. In prokaryotic cephalosporin producers, the cephalosporin product, DAOC, is hydroxylated at the 3′-position to form deacetylcephalosporin C (DAC) as catalysed by a second 2OG-dependent enzyme, DAC synthase (DACS). In eukaryotic cephalosporin producers, the reaction is catalysed by a bifunctional enzyme, DAOC/DACS, that catalyses both the ring expansion and the 3′-hydroxylation reactions. The prokaryotic and eukaryotic enzymes are closely related to DAOCS by sequence, suggesting these enzymes may have evolved by gene duplication. Cephamycin C-producing microorganisms use two enzymes, encoded by the genes cmcI/J, to convert cephalosporins to their 7α-methoxy derivatives that are less vulnerable to β-lactam hydrolysing enzymes. The methoxylation reaction is dependent on Fe(ii), 2OG and S-adenosylmethionine, suggesting the involvement of another 2OG-dependent oxygenase. Herein, structural and mechanistic features are summarized for these 2OG enzymes that utilize this common and flexible mode of dioxygen activation.

Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO(2)/O(2) specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the restrictive temperature identified an Ala222 to Thr (A222T) substitution that suppresses the deleterious effects of the original mutant substitution to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme. Because the mutated residues are situated approximately 19 A away from the active site, they must affect the relative rates of carboxylation and oxygenation in an indirect way. As a means for elucidating the role of such distant interactions in Rubisco catalysis and stability, we have determined the crystal structures of the L290F mutant and L290F/A222T revertant enzymes to 2.30 and 2.05 A resolution, respectively. Inspection of the structures reveals that the mutant residues interact via van der Waals contacts within the same large subunit (intrasubunit path, 15.2 A Calpha-Calpha) and also via a path involving a neighboring small subunit (intersubunit path, 18.7 A Calpha-Calpha). Structural analysis of the mutant enzymes identified regions (residues 50-72 of the small subunit and residues 161-164 and 259-264 of the large subunit) that show significant and systematically increased atomic temperature factors in the L290F mutant enzyme compared to wild type. These regions coincide with residues on the interaction paths between the L290F mutant and A222T suppressor sites and could explain the temperature-conditional phenotype of the L290F mutant strain. This suggests that alterations in subunit interactions will influence protein dynamics and, thereby, affect catalysis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the enzyme assimilating CO2 in biology. Despite serious efforts, using many different methods, a detailed understanding of activity and regulation in Rubisco still eludes us. New results in X-ray crystallography may provide a structural framework on which to base experimental approaches for more detailed analyses of the function of Rubisco at the molecular level. This article gives a critical review of the field and summarizes recent results from structural studies of Rubisco.

The crystal structure of an activated complex of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach and its product 3-phosphoglycerate has been determined to 2.2 A resolution. The structure is of the open form with the active site accessible to the solvent as observed in the structures of the activated ligand-free enzyme and the complex of the activated enzyme with the substrate ribulose-1,5-bisphosphate. Two molecules of 3-phosphoglycerate are bound per active site. The phosphates of both molecules bind approximately at the same position as the phosphates of ribulose-1,5-bisphosphate or the six-carbon intermediate analogue 2-carboxyarabinitol-1,5-bisphosphate, but one product molecule is swung out from the active site with its carboxylate group pointing toward solution. The present structure points to direct participation of the active site side chain of lysine 175 in later stages of catalysis. This possibility is discussed in the light of mutagenesis studies.

The three-dimensional structure of the complex of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco; EC 4.1.1.39) from spinach with its natural substrate ribulose 1,5-bisphosphate (RuBP) has been determined both under activating and non-activating conditions by X-ray crystallography to a resolution of 2.1 A and 2.4 A, respectively. Under activating conditions, the use of calcium instead of magnesium as the activator metal ion enabled us to trap the substrate in a stable complex for crystallographic analysis. Comparison of the structure of the activated and the non-activated RuBP complexes shows a tighter binding for the substrate in the non-activated form of the enzyme, in line with previous solution studies. In the non-activated complex, the substrate triggers isolation of the active site by inducing movements of flexible loop regions of the catalytic subunits. In contrast, in the activated complex the active site remains partly open, probably awaiting the binding of the gaseous substrate. By inspection of the structures and by comparison with other complexes of the enzyme we were able to identify a network of hydrogen bonds that stabilise a closed active site structure during crucial steps in the reaction. The present structure underlines the central role of the carbamylated lysine 201 in both activation and catalysis, and completes available structural information for our proposal on the mechanism of the enzyme.

Glycine decarboxylase, or P-protein, is a pyridoxal 5'-phosphate (PLP)-dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an α2 homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apoenzyme shows the C terminus locked in a closed conformation by a disulfide bond between Cys(972) in the C terminus and Cys(353) located in the active site. The presence of the disulfide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys(972) and Cys(353) by Ser using site-directed mutagenesis have distinctly lower specific activities, supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353-972 disulfide releases the C terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulfide bond that acts to stabilize the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier.

Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/beta-lactamase-type fold with highest structural similarity to the class A beta-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show beta-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, beta-lactamases and d-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the carboxylation of ribulose 1,5-bisphosphate. The reaction catalyzed by Rubisco involves several steps, some of which can occur as partial reactions, forming intermediates that can be isolated. Analogues of these intermediates are potent inhibitors of the enzyme. We have studied the interactions with the enzyme of two inhibitors, xylulose 1,5-bisphosphate and 4-carboxyarabinitol 1,5-bisphosphate, by x-ray crystallography. Crystals of the complexes were formed by cocrystallization under activating conditions. In addition, 4-carboxyarabinitol 1,5-bisphosphate was soaked into preformed activated crystals of the enzyme. The result of these experiments was the release of the activating CO2 molecule as well as the metal ion from the active site when the inhibitors bound to the enzyme. Comparison with the structure of an activated complex of the enzyme indicates that the structural basis for the release of the activator groups is a distortion of the metal binding site due to the different geometry of the C-3 hydroxyl of the inhibitors. Both inhibitors induce closure of active site loops despite the inactivated state of the enzyme. Xylulose 1,5-bisphosphate binds in a hydrated form at the active site.

Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes.

Deacetoxycephalosporin C synthase (DAOCS) is a mononuclear ferrous enzyme, which catalyzes the expansion of the five-membered thiazolidine ring of the penicillin nucleus into the six-membered dihydrothiazine ring of the cephalosporin nucleus. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron–oxygen intermediate (a ferryl or peroxo compound) from a ferrous iron in the active site of DAOCS. The extended family of 2-oxoacid-dependent ferrous enzymes, to which DAOCS belongs, possesses a common jelly roll β-barrel fold with the ferrous iron ligated by two histidines and a carboxylate. Here we describe the high-resolution structures of apo-DAOCS, and of the enzyme in complex with Fe(II) and co-substrate 2-oxoglutarate. On the basis of the structures and the quantum-chemical calculations, a mechanism is proposed whereby the reactive oxidizing species may be stored in a safe form within the enzyme to await the reaction with the penicillin substrate when it is available. 3D Structure Keywords: ring expansion; cephalosporins; mononuclear ferrous oxygenase; ferryl formation; b-lactam antibiotics

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L(2) dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the key first step in photosynthetic CO2 fixation, the reaction that incorporates CO2 into sugar. In this study, refined crystal structures of unactivated tobacco RuBisCO and activated RuBisCO from spinach and tobacco, in complex with the reaction-intermediate analog 2-carboxyarabinitol 1,5-bisphosphate (CABP), are compared. Both plant enzymes are hexadecameric complexes of eight large and eight small subunits with a total relative molecular mass of approximately 550,000. The comparison of activated and unactivated forms of RuBisCO provides insight into the dynamics of action of this enzyme. The catalytic site, which is open to the solvent in the unactivated enzyme, becomes shielded in the activated CABP complex. This shielding is accomplished by a 12-A movement of the active-site "loop 6" (residues 331-338) and a disorder-order transition of three loops near the active-site entrance, the N terminus, the C terminus, and a loop comprising residues 64-68. All these residues belong to the catalytic large subunit. Domain rotations of about 2 degrees are observed, also tightening the active-site cleft. These observations provide an explanation for the extremely tight binding (Kd < or = 10(-11) M) of the CABP molecule. A striking correlation exists between crystallographic temperature factors in the activated enzyme and the magnitude of the atomic movement upon activation.

The X-ray structure of the quaternary complex of ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach with CO2, Mg2+ and a reaction-intermediate analogue (CABP) has been determined and refined at 2·4 A resolution. Cyclic non-crystallographic symmetry averaging around the molecular 4-fold axis and phase combination were used to improve the initial multiple isomorphous replacement phases. A model composed of one large subunit and one small subunit was built in the resulting electron density map, which was of excellent quality. Application of the local symmetry gave an initial model of the L8S8 molecule with a crystallographic R-value of 0·43. Refinement of this initial model was performed by a combination of conventional least-squares energy refinement and molecular dynamics simulation using the XPLOR program. Three rounds of refinement, interspersed with manual rebuilding at the graphics display, resulted in a model containing all of the 123 amino acid residues in the small subunit, and 467 of the 475 residues in the large subunit. The R-value for this model is 0·24, with relatively small deviations from ideal stereochemistry. Subunit interactions in the L8S8 molecule have been analysed and are described. The interface areas between the subunits are extensive, and bury almost half of the accessible surface areas of both the large and the small subunit. A number of conserved interaction areas that may be of functional significance have been identified and are described. The binding of 2-carboxy-arabinitol 1,5-bisphosphate to the active site is described, and biochemical and mutagenesis data are discussed in the structural framework of the model.

The ultimate step in the biosynthesis of the medicinally important beta-lactamase inhibitor clavulanic acid is catalyzed by clavulanic acid dehydrogenase (CAD). CAD is responsible for the NAPDH-dependent reduction of the unstable intermediate clavulanate-9-aldehyde to yield clavulanic acid. Here, we report biochemical and structural studies on CAD. Biophysical analyses demonstrate that CAD exists as dimeric and tetrameric species in solution. The reaction performed by CAD was shown to be reversible, allowing the use of clavulanic acid for activity analyses. The crystal structure of CAD was solved using single-wavelength anomalous diffraction with a seleno-methionine derivative. The structure reveals that the individual monomers comprise a single domain possessing the Rossmann fold, characteristic of dinucleotide-binding enzymes. The monomers are arranged as tetramers, similar to other tetrameric members of the short-chain dehydrogenase/reductase family. The structure of the unreactive complex of CAD with clavulanic acid and NADPH suggests how CAD is able to catalyze the reduction of clavulanate-9-aldehyde without fragmentation of the bicyclic beta-lactam ring structure. The relative positions of NADPH and clavulanic acid, in the active site, together with the presence of the latter in an eclipsed conformation, rationalizes previous labeling studies demonstrating that the incorporation of the C5 pro-R, but not pro-S, hydrogen of ornithine/arginine into the C9 position of clavulanic acid occurs with overall inversion of configuration.

Deacetoxycephalosporin C synthase (DAOCS) is a mononuclear ferrous enzyme that catalyzes the expansion of the five-membered thiazolidine ring of the penicillin nucleus into the six-membered dihydro ...

Cephamycin C-producing microorganisms use two enzymes to convert cephalosporins to their 7alpha-methoxy derivatives. Here we report the X-ray structure of one of these enzymes, CmcI, from Streptomyces clavuligerus. The polypeptide chain of the enzyme folds into a C-terminal Rossmann domain and a smaller N-terminal domain, and the molecule packs as a hexamer in the crystal. The Rossmann domain binds S-adenosyl-L-methionine (SAM) and the demethylated product, S-adenosyl-L-homocysteine, in a fashion similar to the common binding mode of this cofactor in SAM-dependent methyltransferases. There is a magnesium-binding site in the vicinity of the SAM site with a bound magnesium ion ligated by residues Asp160, Glu186 and Asp187. The expected cephalosporin binding site near the magnesium ion is occupied by polyethyleneglycol (PEG) from the crystallisation medium. The geometry of the SAM and the magnesium binding sites is similar to that found in cathechol O-methyltransferase. The results suggest CmcI is a methyltransferase, and its most likely function is to catalyse the transfer of a methyl group from SAM to the 7alpha-hydroxy cephalosporin in the second catalytic reaction of cephamycin formation. Based on the docking of the putative substrate, 7alpha-hydroxy-O-carbamoyldeacetylcephalosporin C, to the structure of the ternary CmcI-Mg2+-SAM complex, we propose a model for substrate binding and catalysis. In this model, the 7-hydroxy group of the beta-lactam ring ligates the Mg2+ with its alpha-side facing the methyl group of SAM at a distance that would allow methylation of the hydroxyl-group.

Background The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in the metabolism of a major plant hormone. The activity of recombinant GA 2ODDs from many species has been characterised in detail, however little information relates to enzyme purification. Native GA 2ODDs displayed lability during purification. Results Two GA 2ODDs were expressed in Escherichia coli and purified to homogeneity. The GA 2-oxidase from Pisum sativum L., PsGA2OX1, was expressed as a glutathione s-transferase (GST) fusion. It was purified in the three steps of affinity chromatography, GST removal and gel filtration. Highly pure PsGA2OX1 was obtained at a yield of 0.3 mg/g of cells. It displayed a K m of 0.024 μM and a V max of 4.4 pkat/mg toward [1β,2β,3β-3H3]GA20. The GA 3-oxidase from Arabidopsis thaliana, AtGA3OX4, was expressed as a poly(His)-tagged thioredoxin fusion. It was purified by Immobilised Metal Affinity Chromatography followed by gel filtration. Cleavage of the fusion took place between the two purification steps. Highly pure AtGA3OX4 was obtained at a yield of 0.01 mg/g of cells. It displayed a K m of 0.82 μM and V max of 52,500 pkat/mg toward [1β,2β,3β-3H3]GA20. Conclusion Fusion tags were required to stabilise and solubilise PsGA2OX1 and AtGA3OX4 during E. coli expression. The successful purification of milligram quantities of PsGA2OX1 enables mechanistic and structural studies not previously possible on GA 2ODDs. A moderate yield of pure AtGA3OX4 requires the further optimisation of the latter stages of the enzyme purification schedule. PsGA2OX1's action in planta as deduced from the effect of the null mutation sln on GA levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.

Cephamycins are broad-spectrum beta-lactam antibiotics that show resistance to certain forms of beta-lactamases. They differ from cephalosporins by the presence of a methoxyl group at the C-7alpha position. The gene products of cmcI and cmcJ are believed to control 7alpha-methoxylation of cephalosporins through successive steps of hydroxylation and methylation. Here, the expression, purification, crystallization and initial data-collection statistics of the 236-amino-acid protein product of cmcI from Streptomyces clavuligerus is reported. The crystals belong to space group P2(1), with unit-cell parameters a = 93.6, b = 182.6, c = 103.2 A, beta = 91.05 degrees. Diffraction data were collected to 2.5 A.

The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 A resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between beta strands A and B of the small subunit (betaA-betaB loop), which is longer by six amino acid residues than the corresponding region in Rubisco from Spinacia. Mutations of residues in the betaA-betaB loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonas structure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.

Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are catalysed by Fe(II)-dependent oxidases/oxygenases; isopenicillin N synthase (IPNS) creates in one step the bicyclic nucleus of penicillins, and deacetoxycephalosporin C synthase (DAOCS) catalyses the expansion of the penicillin nucleus into the nucleus of cephalosporins. Both enzymes use dioxygen-derived ferryl intermediates in catalysis but, in contrast to IPNS, the ferryl form of DAOCS is produced by the oxidative splitting of a co-substrate, 2-oxoglutarate (alpha-ketoglutarate). This route of controlled ferryl formation and reaction is common to many mononuclear ferrous enzymes, which participate in a broader range of reactions than their well-characterized counterparts, the haem enzymes. Here we report the first crystal structure of a 2-oxoacid-dependent oxygenase. High-resolution structures for apo-DAOCS, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate, were obtained from merohedrally twinned crystals. Using a model based on these structures, we propose a mechanism for ferryl formation.

The effect of remoxipride ((S)(-)3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2,6-dimethoxybenzam ide) on dopamine D2 receptor-mediated inhibition of cAMP formation in rat striatal tissues pieces was established together with that of a number of other dopamine D2 receptor antagonists. The action of remoxipride, three other substituted benzamides, (-)-sulpiride, raclopride and NCQ 298 ((S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6-dimethoxysalicylam ide mesylate) and haloperidol, a butyrophenone, was studied in the presence of (I) (+/-) SKF 38393 (7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) hydrochloride (100 microM) plus pergolide (1 microM) or (II) forskolin (1 microM) plus dopamine (100 microM). In addition, four of the metabolites of remoxipride: FLA 797 ((S)-3-bromo-N[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-6- methoxybenzamide), NCR 181 ((S)(-)-5-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-6- methoxybenzamide tartrate), NCQ 436 ((S)(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6-dihydroxy-2- methoxybenzamide semioxalate) and NCQ 469 ((S)(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-hydroxy-2,6- dimethoxybenzamide hydrochloride), mainly found in rodents, were studied using test system I. The results demonstrate that remoxipride is significantly weaker in blocking functional striatal dopamine D2 receptors than either of the reference compounds studied and three of the four metabolites. The studies also demonstrate that dopamine D1 and D2 receptor interactions at the level of cAMP formation in the striatum are independent of action potentials or Ca2+.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has attracted a lot of interest due to its central role in the carbon metabolism of plants and photosynthetic microorganisms (for a review see (1)). The dual function of this enzyme, catalyzing the primary steps in both photosynthetic carbon dioxide fixation and photorespiration (Figure 1), makes it a challenging target for attempts to improve the efficiency of photosynthesis. Recombinant DNA-techniques provide a promising tool to modify the carboxylase/oxygenase ratio by genetic engineering. However, the application of these techniques requires a detailed knowledge of the catalytic mechanism of the enzyme and the structure of its active site.

An electron density map to 2.9 Å (1 Å = 10 -10 m) resolution of Rubisco from Rhodospirillum rubrum has been obtained from crystals of the gene product expressed in Escherichia coli . These crystals are monoclinic, space group P2 1 with cell dimensions a = 65.5 Å, b = 70.6 Å, c = 104.1 Å and β = 92° and with two subunits per asymmetric unit. Isomorphous phases were obtained from three heavy atom derivatives. The dimeric molecule has the shape of a distorted ellipsoid with approximate dimensions 45 x 70 x 105 Å. The subunit interactions in the dimeric molecule are tight and extensive. Each subunit is divided into two main domains, one of which has extensive α/β structure probably folded into the common eight unit barrel structure. NADPH and pyridoxal phosphate bind at one end of this domain in each subunit. Two different octameric molecules with approximate (422) symmetry have been constructed from the L 2 dimeric molecule from R. rubrum , assuming that the molecular twofold axis of the L 2 dimer is preserved in the L 8 molecule. It is shown that only one of these is compatible with the orthorhombic crystal lattice of spinach and Alcaligenes eutrophus Rubisco. The L 8 molecule thus obtained is ellipsoidal with approximate dimensions 130 Å x 130 Å x 105 Å and with a large hole in the middle of about 40 Å diameter around the fourfold axis. A cleft in each subunit between the two domains could form a binding site for the small subunit in the plant L 8 S 8 Rubisco molecule.

The retention behaviour of amines was investigated in systems with silica gel (LiChrosorb Si 60) as the support and an aqueous phosphate buffer containing organic modifiers as the mobile phase. N,N-Dimethyloctylamine and N,N,N-trimethyloctylamine were used as cationic modifiers, with 3,5-dimethylcyclohexylsulphate as the ion-pair forming counter ion. Adsorption isotherms were determined for the amines used as modifiers. At low concentrations of modifier a monolayer is formed on the solid phase and the retention times of the solutes decrease with increasing modifier concentration. Higher concentrations give rise to the formation of a multilayer or bulk phase of modifier, leading to rapidly increased retention of the most hydrophobic solutes and a change in retention order. They hydrogen-bonding properties of the solute seem to be of major importance for the retention on unmodified silica, while the hydrophobicity of the solute determines the retention to the adsorbed phase of the modifier.

The structure of the complex between Cys-46-carboxymethylated horse liver alcohol dehydrogenase (CM-LADH) and reduced nicotinamide adenine dinucleotide (NADH) has been determined by X-ray analysis. The complex represents NADH binding to the orthorhombic, "open" conformation of the enzyme. Coenzyme binding here induces a local structural change in the peptide loop 293-297, but there is no domain rotation, as observed for the "closed" conformation of the protein. This local movement of a few residues in the loop is sufficient to trap the nicotinamide ring of NADH within the active-site area close to a productive binding position. The carboxymethyl group on the zinc ligand cysteine-46 is oriented between the pyrophosphate bridge of NADH and the guanidinium group of arginine-369 and can occupy this position because the coenzyme binding cleft remains open and unchanged upon coenzyme binding. The zinc coordination sphere is distorted, and the position of the metal atom is shifted 1 A compared to native unliganded LADH. The distance between the zinc ion and the sulfur of the alkylated cysteine residue is of the order of 3 A. Alkylation experiments were performed at 0.15 and 10 mM iodoacetate, and peptide maps were examined. Gentle treatment with reagent yields an enzyme product which is substituted at only one of the two zinc binding sites per subunit of LADH (Cys-46). This enzyme species maintains its structural integrity; it binds coenzyme which induces conformational changes resolved into two steps. Thus, in addition to the orthorhombic complex, a crystalline NADH complex in the closed conformation of CM-LADH was obtained. These crystals showed enzymic activity, and single crystals were analyzed with microspectrophotometric methods. Formation of the stable crystalline abortive complex between CM-LADH-NAD+ and 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) could be observed upon addition of excess aldehyde to the closed complex of CM-LADH-NADH. The CM-LADH-NAD+-DACA complex is characterized by an intense absorption band with a lambda max at 456 nm which corresponds to a shift in the spectrum of free DACA of approximately 60 nm. At the higher concentration of iodoacetate, three of the cysteine ligands to the second zinc atom (Cys-100, -103, and -111) are alkylated in addition to Cys-46. This enzyme product rapidly denatures and cannot be crystallized under our conditions. This is an experimental indication that the intact noncatalytic zinc binding site contributes to the structural stability of the protein.

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been purified from spinach and crystallized by equilibrium vapor diffusion with polyethylene glycol 6000 as a precipitant. Crystals suitable for x-ray studies were obtained from a binary complex with a transition state analogue, 2-C-carboxy-D-arabinitol 1,5-bisphosphate, and a quaternary complex with 2-C-carboxy-D-arabinitol 1,5-bisphosphate, Mg2+, and HCO-3. Two forms of crystals were obtained in the presence of 2-C-carboxy-D-arabinitol 1,5-bisphosphate. Form B crystals are plates which have orthorhombic space group P2(1)2(1)2 with unit cell dimensions a = 184 A, b = 218 A, and c = 119 A. Form C crystals are tetragonal needles with space group I422 and with cell dimensions a = b = 275 A and c = 178 A. In both forms, the asymmetric unit contains half a molecule.

The 2-deoxy analogue of 3-deoxy-beta-D-manno-2-octulopyranosonic acid (2-deoxy-beta-KDO, 2) is a potent inhibitor of the enzyme 3-deoxy-D-manno-octulosonate cytidylyltransferase, which is involved in the biosynthesis of lipopolysaccharide, an essential component of the outer membrane of Gram-negative bacteria. Since compound 2 lacks antibacterial activity, a series of 8-substituted derivatives of 2 has been synthesized in an attempt to find enzyme inhibitors suitable for modification as antibacterials. Compounds 9, 11, and 13, in which the 8-hydroxy group of 2 is replaced by F, H, and NH2, respectively, were as potent inhibitors of the enzyme as 2, but were devoid of antibacterial activity, with the exception of the amino acid 13, which showed weak activity against some strains of Salmonella typhimurium.

Ribulose-1, 5-bisphosphate carboxylase/oxygenase, Rubisco, plays an important role in photosynthesis as well as in photorespiration. In photosynthesis, the carboxylase activity of the enzyme catalyzes the condensation of carbon dioxide and ribulose-1, 5-bisphosphate to yield two moles of 3-D-phosphoglycerate (Miziorko and Lorimer, 1983; Andrews and Lorimer, 1987). In addition, Rubisco has an oxygenase activity whereby oxygen is added to ribulose 1, 5-bisphosphate to yield 3-D-phosphoglycerate and 2-phosphoglycolate, the major substrate for photorespiration. As a result of the subsequent metabolism of phosphoglycolate one of the carbon atoms is oxidized to carbon dioxide and the released energy is lost as heat. Up to 50% of the photosynthetically reduced carbon may be oxidized through this pathway. The possibility to increase the carboxylase/oxygenase ratio has therefore attracted substantial interest. An understanding of the structural basis for the two activities would greatly facilitate the successful use of site directed mutagenesis techniques towards this end.

Ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco is bifunctional (Miziorko, Lorimer, 1983). In photosynthesis, the carboxylase activity of the enzyme catalyzes the condensation of CO2 to ribulose 1,5-bisphosphate (RuBP) to yield two moles of 3-D-phosphoglycerate (PGA). The oxygenase activity, on the other hand, catalyzes the addition of O2 to RuBP to yield PGA and 2-phosphoglycolate. The last reaction results in net loss of fixed CO2. An understanding of the structural basis for these two activities is therefore important for genetic modifications of Rubisco in plants (Sommerville, Ogren, 1982).