Structure of the Mg-chelatase cofactor GUN4 reveals a novel hand-shaped fold for porphyrin binding.
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ABSTRACT:
SUBMITTER: Verdecia MA
PROVIDER: S-EPMC1084334 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2--dependent insertion of a Mg2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.
| S-EPMC6604950 | biostudies-literature
Project description:In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of ?6/?7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.
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Project description:Mg-chelatase catalyses the insertion of Mg into protoporphyrin IX (Proto). This seemingly simple reaction also is potentially one of the most interesting and crucial steps in the (bacterio)chlorophyll (Bchl/Chl)-synthesis pathway, owing to its position at the branch-point between haem and Bchl/Chl synthesis. Up until the level of Proto, haem and Bchl/Chl synthesis share a common pathway. However, at the point of metal-ion insertion there are two choices: Mg2+ insertion to make Bchl/Chl (catalysed by Mg-chelatase) or Fe2+ insertion to make haem (catalysed by ferrochelatase). Thus the relative activities of Mg-chelatase and ferrochelatase must be regulated with respect to the organism's requirements for these end products. How is this regulation achieved? For Mg-chelatase, the recent design of an in vitro assay combined with the identification of Bchl-biosynthetic enzyme genes has now made it possible to address this question. In all photosynthetic organisms studied to date, Mg-chelatase is a three-component enzyme, and in several species these proteins have been cloned and expressed in an active form. The reaction takes place in two steps, with an ATP-dependent activation followed by an ATP-dependent chelation step. The activation step may be the key to regulation, although variations in subunit levels during diurnal growth may also play a role in determining the flux through the Bchl/Chl and haem branches of the pathway.
| S-EPMC1218797 | biostudies-other
Project description:To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression.
| S-EPMC4683165 | biostudies-literature
Project description:There is a high functional diversity within the structural superfamily of porphyrin-binding dimeric α + β barrel proteins. In this review we aim to analyze structural constraints of chlorite dismutases, dye-decolorizing peroxidases and coproheme decarboxylases in detail. We identify regions of structural variations within the highly conserved fold, which are most likely crucial for functional specificities. The loop linking the two ferredoxin-like domains within one subunit can be of different sequence lengths and can adopt various structural conformations, consequently defining the shape of the substrate channels and the respective active site architectures. The redox cofactor, heme b or coproheme, is oriented differently in either of the analyzed enzymes. By thoroughly dissecting available structures and discussing all available results in the context of the respective functional mechanisms of each of these redox-active enzymes, we highlight unsolved mechanistic questions in order to spark future research in this field.
| S-EPMC7611857 | biostudies-literature
Project description:The heme biosynthetic pathway culminates with the ferrochelatase-catalyzed ferrous iron chelation into protoporphyrin IX to form protoheme. The catalytic mechanism of ferrochelatase has been proposed to involve the stabilization of a nonplanar porphyrin to present the pyrrole nitrogens to the metal ion substrate. Previously, we hypothesized that the ferrochelatase-induced nonplanar distortions of the porphyrin substrate impose selectivity for the divalent metal ion incorporated into the porphyrin ring and facilitate the release of the metalated porphyrin through its reduced affinity for the enzyme. Using resonance Raman spectroscopy, the structural properties of porphyrins bound to the active site of directly evolved Ni(2+)-chelatase variants are now examined with regard to the mode and extent of porphyrin deformation and related to the catalytic properties of the enzymes. The Ni(2+)-chelatase variants (S143T, F323L, and S143T/F323L), which were directly evolved to exhibit an enhanced Ni(2+)-chelatase activity over that of the parent wild-type ferrochelatase, induced a weaker saddling deformation of the porphyrin substrate. Steady-state kinetic parameters of the evolved variants for Ni(2+)- and Fe(2+)-chelatase activities increased compared to those of wild-type ferrochelatase. In particular, the reduced porphyrin saddling deformation correlated with increased catalytic efficiency toward the metal ion substrate (Ni(2+) or Fe(2+)). The results lead us to propose that the decrease in the induced protoporphyrin IX saddling mode is associated with a less stringent metal ion preference by ferrochelatase and a slower porphyrin chelation step.
| S-EPMC3050429 | biostudies-literature
Project description:Activation of p53 target genes for tumor suppression depends on the stress-specific regulation of transcriptional coactivator complexes. Strap (stress-responsive activator of p300) is activated upon DNA damage by ataxia telangiectasia mutated (ATM) and Chk2 kinases and is a key regulator of the p53 response. In addition to antagonizing Mdm2, Strap facilitates the recruitment of p53 coactivators, including JMY and p300. Strap is a predicted TPR-repeat protein, but shows only limited sequence identity with any protein of known structure. To address this and to elucidate the molecular mechanism of Strap activity we determined the crystal structure of the full-length protein at 2.05 Å resolution. The structure of Strap reveals an atypical six tetratricopeptide repeat (TPR) protein that also contains an unexpected oligonucleotide/oligosaccharide-binding (OB)-fold domain. This previously unseen domain organization provides an extended superhelical scaffold allowing for protein-protein as well as protein-DNA interaction. We show that both of the TPR and OB-fold domains localize to the chromatin of p53 target genes and exhibit intrinsic regulatory activity necessary for the Strap-dependent p53 response.
| S-EPMC3309724 | biostudies-literature
Project description:The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.
| S-EPMC155376 | biostudies-literature
Project description:As a rate-limiting enzyme for chlorophyll biosynthesis, Mg-chelatase is a promising target for improving photosynthetic efficiency. It consists of CHLH, CHLD, and CHLI subunits. In pea (Pisum sativum L.), two putative CHLI genes (PsCHLI1 and PsCHLI2) were revealed recently by the whole genome sequencing, but their molecular features are not fully characterized. In this study, PsCHLI1 and PsCHLI2 cDNAs were identified by PCR-based cloning and sequencing. Phylogenetic analysis showed that PsCHLIs were derived from an ancient duplication in legumes. Both PsCHLIs were more highly expressed in leaves than in other organs and downregulated by abscisic acid and heat treatments, while PsCHLI1 was more highly expressed than PsCHLI2. PsCHLI1 and PsCHLI2 encode 422- and 417-amino acid proteins, respectively, which shared 82% amino acid identity and were located in chloroplasts. Plants with a silenced PsCHLI1 closely resembled PsCHLI1 and PsCHLI2 double-silenced plants, as both exhibited yellow leaves with barely detectable Mg-chelatase activity and chlorophyll content. Furthermore, plants with a silenced PsCHLI2 showed no obvious phenotype. In addition, the N-terminal fragment of PsCHLI1 (PsCHLI1N, Val63-Cys191) and the middle fragment of PsCHLI1 (PsCHLI1M, Gly192-Ser336) mediated the formation of homodimers and the interaction with CHLD, respectively, while active PsCHLI1 was only achieved by combining PsCHLI1N, PsCHLI1M, and the C-terminal fragment of PsCHLI1 (Ser337-Ser422). Taken together, PsCHLI1 is the key CHLI subunit, and its peptide fragments are essential for maintaining Mg-chelatase activity, which can be used to improve photosynthetic efficiency by manipulating Mg-chelatase in pea.
| S-EPMC8821089 | biostudies-literature
Project description:The pyridoxal-P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal-P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo- and holoenzymes. PROBE identified six motifs shared by the two GADs and four proteins of known structure: bacterial ornithine decarboxylase, dialkylglycine decarboxylase, aspartate aminotransferase, and tyrosine phenol-lyase. Five of the motifs corresponded to the alpha/beta elements and loops that form most of the conserved fold of the pyridoxal-P binding cleft of the four enzymes of known structure; the sixth motif corresponded to a helical element of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal-P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functions were evaluated by examining the proteins of known structure. The key residues that interact directly with pyridoxal-P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a specific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates that the highly regulated transition between apo- and holoGAD is accomplished by modifications in this basic fold rather than through a novel folding pattern.
| S-EPMC2144009 | biostudies-other