Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, α and β. The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the α and β subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments. In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure. However, in the presence of MgATP, the β subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, α subunits only assemble in the presence of β subunits. Although β and α/β 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different. Similar to the authentic chloroplast protein, the reconstituted α/β 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10. In contrast, the folding reaction mediated by the reconstituted β 14-mers is only efficient with mitochondrial cpn10. The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system.

Dethiobiotin synthetase (DTBS; E.C. 6.6.6.6), thepenultimate enzyme in the biosynthesis of the essential vitaminbiotin, is a new potential target for novel herbicides. Inhibitorswere designed based on mechanistic and structural information. Thein-vitro activities of these potential inhibitors versus thebacterial enzyme are reported here. Mimics of7,8-diaminopelargonic acid (DAPA) or the DAPAcarbamate reaction intermediate were substrates or partial substratesfor the enzyme. Synergistic binding with ATP was noted with compoundswhich contained an amino functionality. NMR studies and X-raystructures confirmed that the inhibitors could be phosphorylated bythe enzyme. Several series of potential inhibitors were designed totake advantage of this partial substrate activity by generatingpotentially more tightly bound phosphorylated inhibitors in situ. Structure–activity relationships for these series based onboth substrate and inhibitory activity are described herein. AnX-ray structure for one of these inhibitors is also discussed.Although considerable potential for inhibitors of this type wasdemonstrated, none of the compounds reported showed sufficientherbicidal activity to be a commercial proposition. © 1999 Society of Chemical Industry

Background: In Escherichia coli, the enzymes of the biotin biosynthesis pathway are encoded by the bio operon. One of these enzymes, ATP-dependent dethiobiotin synthetase, catalyzes the carboxylation of 7,8-diaminopelargonic acid leading to the formation of the ureido ring of biotin. The enzyme belongs to the class of ATP-dependent carboxylases and we present here the first crystal structure determined for this class of enzyme.Results: We have determined the crystal structure of homodimeric dethiobiotin synthetase to 1.65 angstrom resolution. The subunit consists of a seven-stranded parallel beta-sheet, surrounded by alpha-helices. The sheet contains the classical mononucleotide-binding motif with a fingerprint peptide Gly-X-X-X-X-X-Gly-Lys-Thr. The mononucleotide binding part of the structure is very similar to the GTP-binding protein H-ras-p21 and thus all GTP-binding proteins. A comparison reveals that some of the residues, which in H-ras-p21 interact with the nucleotide and the metal ion, are conserved in the synthetase.Conclusions: The three-dimensional structure of dethiobiotin synthetase has revealed that ATP-dependent carboxylases contain the classical mononucleotide-binding fold. Considerable similarities to the structure of the GTP-binding protein H-ras-p21 were found, indicating that both proteins might have evolved from a common ancestral mononucleotide-binding fold.

The particular structural arrangement of chaperonins probably contributes to their ability to assist in the folding of proteins. The interaction of the oligomeric bacterial chaperonin GroEL and its cochaperonin, GroES, in the presence of adenosine diphosphate (ADP) forms an asymmetric complex. However, in the presence of adenosine triphosphate (ATP) or its nonhydrolyzable analogs, symmetric complexes were found by electron microscopy and image analysis. The existence of symmetric chaperonin complexes is not predicted by current models of the functional cycle for GroE-mediated protein folding. Because complete folding of a nonnative substrate protein in the presence of GroEL and GroES only occurs in the presence of ATP, but not with ADP, the symmetric chaperonin complexes formed during the GroE cycle are proposed to be functionally significant.

Crystals of the hexadecameric form of ribulose-bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra-dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site-directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose-bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.

Truncations of the subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum were generated by site‐directed mutagenesis to examine the role of the C‐terminal tail section. Removal of the last and the penultimate alpha‐helices in the tail section changes the quaternary structure of the protein. Electrophoretic and electron microscope analysis revealed that the truncated subunits assemble into an octamer, whereas the wild‐type enzyme has a dimeric structure. The octomerization of the mutant protein is due to a hydrophobic patch exposed to the solvent by truncation of the subunit. The mutant protein thus consists of four dimers, bound end‐to‐end by hydrophobic interactions. Insertion of a polar amino acid in the hydrophobic patch by a L424 to N424 substitution restores the familiar dimeric structure. Truncation of the subunit is associated with a considerable decrease in catalytic activity. The mutants undergo carbamylation but bind the reaction intermediate analog, 2‐carboxy arabinitol‐1,5‐bisphosphate, poorly. This indicates that loss of activity in the mutant is due to weakened substrate binding. These findings suggest that the mutations in the tail section of the subunit are transmitted to the active site, although the C‐terminal region is far from the active site. On the basis of the crystal structure of Rubisco, we propose a model for how the truncations of the enzyme subunit induce conformational changes in one of the two phosphate binding sites.

Recent progress in in vitro genetic manipulations (1), and in the structural analysis of Rubisco (2,3) have provided the basis for a rationale mutagenesis of this key enzyme in the photosynthetic carbon metabolism, in attempts to define structure/function relationships. In combination with a better knowledge in the chemistry of the enzymatic reactions, studies of specific changes of highly conserved residues within the active site have been developed. So far, the role of at least two residues essential for activation (Lys 191 in Rhodospirillum rubrum Rubisco) or catalysis (Lys 166) have been defined (4,5). Another strategy consists to examine the functional importance of peptide regions of low homology. Construction of chimaeric genes by sequence replacement have indicated the critical requirement of some regions of the large subunit (i.e. N-terminus, bridge region between N- and C-terminal domains) for the assembly and/or function of the protein (6,7). A similar approach was used here to investigate the role of the C-terminus of the large subunit. Sequence deletion in the tail domain was performed on the gene coding for R.rubrum Rubisco. Mutation was designed to remove the last and pen-ultimate α-helices from the C-terminal extension (Fig.l).

2'-Carboxy-D-arabinitol 1-phosphate (2CA1P), a natural inhibitor of ribulose 1,5-bisphosphate carboxylase was synthesized from 2'-carboxy-D-arabinitol 1,5-bisphosphate (2CABP). The selective dephosphorylation of 2CABP with either acid phosphatase or alkaline phosphatase was investigated by using 31P n.m.r. The n.m.r. spectra of the progress of the reactions indicated that both phosphatases preferentially removed the 5-phosphate from the bisphosphate. After the consumption of all of the bisphosphate, alkaline phosphatase generated a mixture of 2'-carboxy-D-arabinitol 1- and 5-monophosphates in the ratio of about 4:1, along with Pi. The enzyme also hydrolysed the monophosphates to 2'-carboxyarabinitol, thus decreasing the yield of 2CA1P further. In contrast, acid phosphatase catalysed almost quantitative conversion of 2CABP into 2CA1P, preferring to hydrolyse only the 5-phosphate. In either case, separation of the 2CA1P from Pi or other products of enzymic hydrolysis was readily accomplished by conventional ion-exchange chromatography or h.p.l.c.

Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme in photosynthetic carbon dioxide fixation and photorespiration. The dimeric carboxylase from the photosynthetic bacterium Rhodospirillum rubrum has been cloned and expressed in E. coli. The recombinant enzyme has been crystallized in a number of different crystal forms. The three-dimensional structure of the enzyme has been determined by X-ray crystallographic methods to 2.9Åresolution.

Photorespiration, which occurs in most photosynthetic organisms, involves an oxidation of reduced carbon in the form of sugar molecules to carbon dioxide. The energy released in this process is dissipated as heat. No net ATP or NADH is produced in this series of enzymatic reactions. Photorespiration is a major process in the carbon metabolism of plants; up to 50% of the solar energy that is absorbed, converted, and stored as reduced carbon is released again by this process with no apparent benefit for the plant.

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.

The three‐dimensional structure of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X‐ray crystallographic methods. The MIR‐electron density map was substantially improved by two‐fold non‐crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two‐domain protein. The smaller, N‐terminal domain consists of 137 amino acid residues and forms a central, mixed five‐stranded β‐sheet with α‐helices on both sides of the sheet. The larger C‐terminal domain consists of 329 amino acid residues. This domain has an eight‐stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non‐related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy‐end of the β‐strands in the α/β barrel of the C‐terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C‐terminal domains, which build up the core of the molecule. There are also interactions between the N‐terminal domain of one subunit and the C‐terminal domain of the second subunit, close to the active site.

Conference Article| June 01 1985 Nature of the activation and active site of ribulose bisphosphate carboxylase from the electron-paramagnetic-resonance transition state enzyme-Mn complexes S. GUTTERIDGE; S. GUTTERIDGE *Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, U.K. Search for other works by this author on: This Site PubMed Google Scholar M. PARRY; M. PARRY *Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, U.K. Search for other works by this author on: This Site PubMed Google Scholar G. SCHMIDT; G. SCHMIDT *Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, U.K. Search for other works by this author on: This Site PubMed Google Scholar G. LORIMER G. LORIMER †C.R. and D., Experimental Station, E.I. du Pont de Nemours Co., Wilmington, DE 19898, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1985) 13 (3): 629–631. https://doi.org/10.1042/bst0130629 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation S. GUTTERIDGE, M. PARRY, G. SCHMIDT, G. LORIMER; Nature of the activation and active site of ribulose bisphosphate carboxylase from the electron-paramagnetic-resonance transition state enzyme-Mn complexes. Biochem Soc Trans 1 June 1985; 13 (3): 629–631. doi: https://doi.org/10.1042/bst0130629 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: ribulose-P2, ribulose bisphosphate, 2CABP, 2-carboxyarabinitol bisphospate, 4CABP, 4-carboxyarabinitol bisphosphate This content is only available as a PDF. © 1985 Biochemical Society1985 Article PDF first page preview Close Modal You do not currently have access to this content.

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate‐divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn2+.2‐carboxyarabinitol 1,5‐bisphosphate and enzyme.activator carbamate.Mn2+.4‐carboxyarabinitol 1,5‐bisphosphate for mutant and wild‐type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.

Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.