| 1. |
- Hasse, Dirk, et al.
(författare)
-
Structure and mechanism of piperideine-6-carboxylate dehydrogenase from Streptomyces clavuligerus
- 2019
-
Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 75, s. 1107-1118
-
Tidskriftsartikel (refereegranskat)abstract
- The core of beta-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. beta-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid alpha-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form alpha-aminoadipate semialdehyde) to alpha-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 from S. clavuligerus in its apo form and in complexes with the cofactor NAD(+), the product alpha-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.
|
|
| 2. |
- Valegård, Karin, et al.
(författare)
-
Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications
- 2018
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 293:34, s. 13033-13043
-
Tidskriftsartikel (refereegranskat)abstract
- The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO2-fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL, and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The V-max and K-m for the oxygenase and carboxylase activities at 25 degrees C and the specificity factors (S-c/o) at 15, 25, and 35 degrees C were determined. The S-c/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO(2 )relative to O-2. Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short beta A-beta B loop and a C-terminal extension that forms a beta-hairpin structure (beta E-beta F loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, beta-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO2 fixation in arctic diatom Rubiscos.
|
|
| 3. |
- Valegård, Karin, et al.
(författare)
-
Structure of Rubisco from Arabidopsis thaliana in complex with 2-carboxyarabinitol-1,5-bisphosphate
- 2018
-
Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 74:1, s. 1-9
-
Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Arabidopsis thaliana is reported at 1.5 Å resolution. In light of the importance of A. thaliana as a model organism for understanding higher plant biology, and the pivotal role of Rubisco in photosynthetic carbon assimilation, there has been a notable absence of an A. thaliana Rubisco crystal structure. A. thaliana Rubisco is an L8S8 hexadecamer comprising eight plastome-encoded catalytic large (L) subunits and eight nuclear-encoded small (S) subunits. A. thaliana produces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B), and this crystal structure provides a snapshot of A. thaliana Rubisco containing the low-abundance RbcS3B small-subunit isoform. Crystals were obtained in the presence of the transition-state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate. A. thaliana Rubisco shares the overall fold characteristic of higher plant Rubiscos, but exhibits an interesting disparity between sequence and structural relatedness to other Rubisco isoforms. These results provide the structural framework to understand A. thaliana Rubisco and the potential catalytic differences that could be conferred by alternative A. thaliana Rubisco small-subunit isoforms.
|
|
| 4. |
- Larsson, Anna M., et al.
(författare)
-
Crystal structures of β-carboxysome shell protein CcmP : ligand binding correlates with the closed or open central pore
- 2017
-
Ingår i: Journal of Experimental Botany. - : OXFORD UNIV PRESS. - 0022-0957 .- 1460-2431. ; 68:14, s. 3857-3867
-
Tidskriftsartikel (refereegranskat)abstract
- 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 beta-carboxysome shell protein CcmP has been shown to form a double layer of pseudohexamers with a relatively large central pore (similar to 13 angstrom 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 angstrom and 1.65 angstrom 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.
|
|
| 5. |
|
|
| 6. |
- Valegård, Karin, et al.
(författare)
-
Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway
- 2013
-
Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69, s. 1567-1579
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 7. |
- Mackenzie, Alasdair, et al.
(författare)
-
Crystal structures of an oligopeptide-binding protein from the biosynthetic pathway of the beta-lactamase inhibitor clavulanic acid.
- 2010
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 396:2
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 8. |
- Cicero, Giancarlo, et al.
(författare)
-
Study of the oxidative half-reaction catalyzed by a non-heme ferrous catalytic center by means of structural and computational methodologies
- 2007
-
Ingår i: International Journal of Quantum Chemistry. - : Wiley. - 0020-7608 .- 1097-461X. ; 107:6, s. 1514-1522
-
Tidskriftsartikel (refereegranskat)abstract
- 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 dihydrothiazine ring of the cephalosporins. In the first half-reaction with dioxygen and 2-oxoglutarate, a reactive iron-oxygen species is produced that can subsequently react with the penicillin substrate to yield the cephalosporin. We describe quantum mechanical calculations of the first part of the reaction based on the high-resolution structures of the active site of DAOCS and its complexes with ligands. These studies are aimed at understanding how the reactive species can be produced and contained in the active site of the enzyme. The results demonstrate the priming of the active site by the co-substrate for oxygen binding and hint to the presence of a stable iron-peroxo intermediate in equilibrium with a more reactive ferryl species and the formation of CO2 as a leaving group by decarboxylation of 2-oxoglutarate. A conclusion from these studies is that substitution of CO2 by the penicillin substrate triggers the oxidation reaction in a booby-trap-like mechanism.
|
|
