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Close-up view of the cofactor-binding sites. (a) Residues interacting with the cofactor. The corresponding 2F o À F c electron-density maps of the cofactor and Lys274 are shown and contoured at 1.0. The cofactor in subunit A is PMP. The cofactor in subunit B is a mixture of PMP and PLP. Lys274 has multiple conformations in each monomer. (b) Interactions between Lys274 and the cofactor, Trp68 and Tyr306*. Hydrogen bonds are depicted as black dotted lines. Distances between the N atom of Lys274 and the C-4 0 atom of the cofactor are depicted as red dotted lines. Distances in A ˚ are displayed in red. The asterisk indicates the residue from the neighbouring subunit.
Source publication
Glutamate-1-semialdehyde-2,1-aminomutase (GSAM) catalyzes the isomerization of glutamate-1-semialdehyde (GSA) to 5-aminolevulinate (ALA) and is distributed in archaea, most bacteria and plants. Although structures of GSAM from archaea and bacteria have been resolved, a GSAM structure from a higher plant is not available, preventing further structur...
Contexts in source publication
Context 1
... agreement with the results of spectral analysis, the AtGSA1 structure displays asymmetry in cofactor binding (Fig. 4). In the OMIT map of subunit A there is continuous electron density between the cofactor and Lys274. However, when PLP is modelled in the ligand density, the distance (2.6 A ˚ ) is not short enough to form a Schiff-base linkage between Lys274 and the cofactor (between the N atom of the "-amino group of Lys274 and the C-4 0 atom of the ...
Context 2
... electron density between the cofactor and Lys274. However, when PLP is modelled in the ligand density, the distance (2.6 A ˚ ) is not short enough to form a Schiff-base linkage between Lys274 and the cofactor (between the N atom of the "-amino group of Lys274 and the C-4 0 atom of the cofactor), demonstrating that the cofactor in subunit A is PMP (Fig. 4a). However, the PMP orientation is different from that previously observed in the PMP-containing subunit of Syne- chococcus GSAM or aspartate aminotransferase, in which the PMP cofactor is usually tilted by 20-30 , moving the amino group away from the catalytic lysine ( Hennig et al., 1997;Jansonius & Vincent, 1987;Stetefeld et al., ...
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... is usually tilted by 20-30 , moving the amino group away from the catalytic lysine ( Hennig et al., 1997;Jansonius & Vincent, 1987;Stetefeld et al., 2006). Instead, the orientation of PMP in subunit A is similar to that of PLP, as research communications previously, with the amino group pointing towards the side chain of the active-site lysine ( Fig. 4; Hennig et al., 1997;Stetefeld et al., 2006). Thus, the continuous electron density between PMP and Lys274 may be owing to the amino group of PMP and the side chain of Lys274 (in one of its multiple conformations) pointing towards each other. The PMP is recognized via hydrogen bonds to Gly124, Thr125, Tyr151, Asn218, Asp246 and Thr306* ...
Context 4
... continuous electron density between PMP and Lys274 may be owing to the amino group of PMP and the side chain of Lys274 (in one of its multiple conformations) pointing towards each other. The PMP is recognized via hydrogen bonds to Gly124, Thr125, Tyr151, Asn218, Asp246 and Thr306* (the asterisk indicates a residue from the neighbouring subunit; Fig. ...
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... emerges and the distance (1.4 A ˚ ) is appropriate for covalent-bond formation between the cofactor and Lys274. Therefore, both PMP and PLP are modelled in the ligand density with occu- pancies of 0.54 and 0.46, respectively. The amino group of PMP points away from Lys274 and PLP forms a Schiff-base linkage with the "-amino group of Lys274 (Fig. 4a), similar to that previously reported in the Synechococcus GSAM struc- ture ( Hennig et al., 1997;Stetefeld et al., 2006). The side chain of Lys274 has three conformations in each subunit: (i) inter- acting with Trp68 and Thr306*, (ii) interacting with PMP by hydrogen bonds in the PMP form and (iii) covalently binding to the cofactor in ...
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... amino group of PMP points away from Lys274 and PLP forms a Schiff-base linkage with the "-amino group of Lys274 (Fig. 4a), similar to that previously reported in the Synechococcus GSAM struc- ture ( Hennig et al., 1997;Stetefeld et al., 2006). The side chain of Lys274 has three conformations in each subunit: (i) inter- acting with Trp68 and Thr306*, (ii) interacting with PMP by hydrogen bonds in the PMP form and (iii) covalently binding to the cofactor in the PLP form (Fig. 4b). Except for Lys274, the residues involved in cofactor fixation in subunit B are similar to those in subunit A (Fig. 4a). ...
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... The side chain of Lys274 has three conformations in each subunit: (i) inter- acting with Trp68 and Thr306*, (ii) interacting with PMP by hydrogen bonds in the PMP form and (iii) covalently binding to the cofactor in the PLP form (Fig. 4b). Except for Lys274, the residues involved in cofactor fixation in subunit B are similar to those in subunit A (Fig. ...
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... fixation has not previously been observed (Fig. 6). As shown in the AtGSA1 structure, subunit A only binds PMP and the gating loop is fixed in the open state, consistent with previous reports that the catalytic reaction is initiated by PMP ( Stetefeld et al., 2006). As the orientation of PMP in subunit A is similar to that of PLP in subunit B (Fig. 4), it is possible that subunit A of AtGSA1 is in the state (Fig. 1, the end of step 6) where PMP has just been regenerated in order to restart the ...
Citations
... The next identified protein was Glutamate-1-semialdehyde 2,1-aminomutase (GSAM; EC 5.4.3.8). This is a widespread enzyme found in prokaryotes (archaea, bacteria) and eukaryotes (plants), catalyzing the glutamate-1-semialdehyde to 5-aminolevulinate conversion (Song et al., 2016). The 5-aminolevulinate is a precursor molecule in the synthesis of tetrapyrrolic groups (hemes), indispensable chemical moieties for biological processes, including photosynthesis and respiration (Song et al., 2016). ...
... This is a widespread enzyme found in prokaryotes (archaea, bacteria) and eukaryotes (plants), catalyzing the glutamate-1-semialdehyde to 5-aminolevulinate conversion (Song et al., 2016). The 5-aminolevulinate is a precursor molecule in the synthesis of tetrapyrrolic groups (hemes), indispensable chemical moieties for biological processes, including photosynthesis and respiration (Song et al., 2016). Heme groups are integral components of different electron transport chain proteins, as well as enzymatic cofactors. ...
Methyl parathion is an organophosphorus pesticide widely employed worldwide to control pests in agricultural and domestic environments. However, due to its intensive use, high toxicity, and environmental persistence, methyl parathion is recognized as an important ecosystem and human health threat, causing severe environmental pollution events and numerous human poisoning and deaths each year. Therefore, identifying and characterizing microorganisms capable of fully degrading methyl parathion and its degradation metabolites is a crucial environmental task for the bioremediation of pesticide-polluted sites. Burkholderia zhejiangensis CEIB S4–3 is a bacterial strain isolated from agricultural soils capable of immediately hydrolyzing methyl parathion at a concentration of 50 mg/L and degrading the 100% of the released p-nitrophenol in a 12-hour lapse when cultured in minimal salt medium. In this study, a comparative proteomic analysis was conducted in the presence and absence of methyl parathion to evaluate the biological mechanisms implicated in the methyl parathion biodegradation and resistance by the strain B. zhejiangensis CEIB S4–3. In each treatment, the changes in the protein expression patterns were evaluated at three sampling times, zero, three, and nine hours through the use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the differentially expressed proteins were identified by mass spectrometry (MALDI-TOF). The proteomic analysis allowed the identification of 72 proteins with differential expression, 35 proteins in the absence of the pesticide, and 37 proteins in the experimental condition in the presence of methyl parathion. The identified proteins are involved in different metabolic processes such as the carbohydrate and amino acids metabolism, carbon metabolism and energy production, fatty acids β-oxidation, and the aromatic compounds catabolism, including enzymes of the both p-nitrophenol degradation pathways (Hydroquinone dioxygenase and Hydroxyquinol 1,2 dioxygenase), as well as the overexpression of proteins implicated in cellular damage defense mechanisms such as the response and protection of the oxidative stress, reactive oxygen species defense, detoxification of xenobiotics, and DNA repair processes. According to these data, B. zhejiangensis CEIB S4–3 overexpress different proteins related to aromatic compounds catabolism and with the PNP degradation pathways, the higher expression levels observed in the two subunits of the enzyme Hydroxyquinol 1,2 dioxygenase, suggest a preferential use of the Hydroquinone metabolic pathway in the p-nitrophenol degradation process. Moreover the overexpression of several proteins implicated in the oxidative stress response, xenobiotics detoxification, and DNA damage repair reveals the mechanisms employed by B. zhejiangensis CEIB S4–3 to counteract the adverse effects caused by the methyl parathion and p-nitrophenol exposure.
... uniprot.org/uniprot/Q6YZE2), which was widely found in photosynthetic algae and plants (Song et al. 2016). In higher plants, the GSAM protein was first purified from the de-etiolated barley (Hordeum vulgare) seedings (Grimm et al. 1989), and GSAM genes have been successively identified from many species, such as soybean (Glycine max, Sangwan and O'Brian 1993), Arabidopsis (Song et al. 2016), tobacco (Nicotiana tabacum, Höfgen et al. 1994;PöRs et al. 2001), tomato (Lycopersicon esculentum, Polking et al. 1995;Lytovchenko et al. 2011), and Brassica napus (Tsang et al. 2003a). ...
... which was widely found in photosynthetic algae and plants (Song et al. 2016). In higher plants, the GSAM protein was first purified from the de-etiolated barley (Hordeum vulgare) seedings (Grimm et al. 1989), and GSAM genes have been successively identified from many species, such as soybean (Glycine max, Sangwan and O'Brian 1993), Arabidopsis (Song et al. 2016), tobacco (Nicotiana tabacum, Höfgen et al. 1994;PöRs et al. 2001), tomato (Lycopersicon esculentum, Polking et al. 1995;Lytovchenko et al. 2011), and Brassica napus (Tsang et al. 2003a). However, no mutant of GSAM has been identified in monocotyledonous plants. ...
Background
Tetrapyrroles play indispensable roles in various biological processes. In higher plants, glutamate 1-semialdehyde 2,1-aminomutase (GSAM) converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA), which is the rate-limiting step of tetrapyrrole biosynthesis. Up to now, GSAM genes have been successively identified from many species. Besides, it was found that GSAM could form a dimeric protein with itself by x-ray crystallography. However, no mutant of GSAM has been identified in monocotyledonous plants, and no experiment on interaction of GSAM protein with itself has been reported so far.
Result
We isolated a yellow leaf mutant, ys53 , in rice ( Oryza sativa ). The mutant showed decreased photosynthetic pigment contents, suppressed chloroplast development, and reduced photosynthetic capacity. In consequence, its major agronomic traits were significantly affected. Map-based cloning revealed that the candidate gene was LOC_Os08g41990 encoding GSAM protein. In ys53 mutant, a single nucleotide substitution in this gene caused an amino acid change in the encoded protein, so its ALA-synthesis ability was significantly reduced and GSA was massively accumulated. Complementation assays suggested the mutant phenotype of ys53 could be rescued by introducing wild-type Os GSAM gene, confirming that the point mutation in OsGSAM is the cause of the mutant phenotype. OsGSAM is mainly expressed in green tissues, and its encoded protein is localized to chloroplast. qRT-PCR analysis indicated that the mutation of OsGSAM not only affected the expressions of tetrapyrrole biosynthetic genes, but also influenced those of photosynthetic genes in rice. In addition, the yeast two-hybrid experiment showed that OsGSAM protein could interact with itself, which could largely depend on the two specific regions containing the 81th–160th and the 321th–400th amino acid residues at its N- and C-terminals, respectively.
Conclusions
We successfully characterized rice GSAM gene by a yellow leaf mutant and map-based cloning approach. Meanwhile, we verified that OsGSAM protein could interact with itself mainly by means of the two specific regions of amino acid residues at its N- and C-terminals, respectively.
... Like a previous crystal structure of a cyanobacterial homolog (Hennig et al. 1997), AtG-SAAT1 forms an asymmetric dimer and displays asymmetry in cofactor binding. While one monomer binds PMP with the gating loop fixed in the open state, the other monomer binds either pyridoxamine phosphate (PMP) or pyridoxal phosphate (PLP) and the gating loop is poised to close (Song et al. 2016). ...
Plastids are specialized organelles found in plants, which are endowed with their own genomes, and differ in many respects from the intracellular compartments of organisms belonging to other kingdoms of life. They differentiate into diverse, plant organ-specific variants, and are perhaps the most versatile organelles known. Chloroplasts are the green plastids in the leaves and stems of plants, whose primary function is photosynthesis. In response to environmental changes, chloroplasts use several mechanisms to coordinate their photosynthetic activities with nuclear gene expression and other metabolic pathways. Here, we focus on a redox-based regulatory network composed of thioredoxins (TRX) and TRX-like proteins. Among multiple redox-controlled metabolic activities in chloroplasts, tetrapyrrole biosynthesis is particularly rich in TRX-dependent enzymes. This review summarizes the effects of plastid-localized reductants on several enzymes of this pathway, which have been shown to undergo dithiol-disulfide transitions. We describe the impact of TRX-dependent control on the activity, stability and interactions of these enzymes, and assess its contribution to the provision of adequate supplies of metabolic intermediates in the face of diurnal and more rapid and transient changes in light levels and other environmental factors.
... Phylogenetic analysis of a family determines how the family might have been derived during evolution. Phylogenetic analysis have reported on Chl synthesis pathway related genes such as GluTRs from higher plants and cyanobacteria (Vasileuskaya et al., 2005), GSA from Arabidopsis thaliana and bacteria (Song et al., 2016). Previous investigation on molecular phylogenetic analysis suggested that plant and bacterial ALA dehydratases (ALAD) have a common lineage not shared by animals or yeast (Kaczor et al., 1994). ...
Chlorophyll (Chl) biosynthesis is one of the most important cellular processes essential for plant photosynthesis. Chl degradation pathway is also important catabolic process occurs during leaf senescence, fruit ripening and under biotic or abiotic stress conditions. Here we have systematically investigated the molecular evolution, gene structure, compositional analysis along with ENc plot, correspondence analysis and codon usage bias of the proteins and encoded genes involved in Chl metabolism from monocots and dicots. The gene and species specific phylogenetic trees using amino acid sequences showed clear clustering formation of the selected species based on monocots and dicots but not supported by 18S rRNA. Nucleotide composition of the encoding genes showed that average GC%, GC1%, GC2% and GC3% were higher in monocots. RSCU analysis depicts that genes from monocots for both pathways and genes for synthesis pathway from dicots only biased to G/C-ending synonymous codons but in degradation pathway most optimal codons (except UUG) in dicots biased to A/U-ending synonymous codons. We found strong evidence of episodic diversifying selection at several amino acid sites in all genes investigated. Conserved domain and gene structures were observed for the genes with varying lengths of introns and exons, involved in Chl metabolism along with some intronless genes within synthesis pathway. ENc and correspondence analyses suggested the mutational or selection constraint on the genes to shape the codon usage. These comprehensive studies may be helpful in further research in molecular phylogenetics and genomics and to better understand the evolutionary dynamics of Chl metabolic pathway.
Communicated by Ramaswamy H. Sarma
... The catalyzing reaction starts from an amino group being transferred from PMP to the substrate GSA when a diamino intermediate and pyridoxal phosphate (PLP) is formed. In a second step, the original amino group of GSA is transferred to PLP, resulting in the regeneration of PMP and the formation of ALA (Song et al., 2016). In Arabidopsis, there are two genes encoding GSAT, GSAT1(AT5G63570), and GSAT2 (AT3G48730). ...
... The crystal structure of GSAT1 from Synechococcus sp. PCC 6301 revealed that GSAT1 forms an asymmetric homodimer with the cofactor (Hennig et al., 1997;Song et al., 2016). From the proposed structure of GluTR-GSAT tetramer, an intermolecular channel for direct transfer of GSA to the active site of GSAT is formed to avoid the release of the highly reactive aldehyde group of GSA (Moser et al., 2001). ...
Fluorescence in blue light (FLU) ist ein negativer Feedbackregulator der Chlorophyllbiosynthese, welcher an der Dunkelrepression der 5-Aminolävulinsäure (ALA)-Synthese beteiligt ist. FLU ist Teil eines Komplexes, der die Enzyme umfasst, welche an der Katalyse der finalen Schritte der Chlorophyllbiosynthese beteiligt sind. Drei funktionelle Domänen wurden für das Arabidopsis FLU Protein postuliert: eine Tetratricopeptid-Wiederholungsdomäne (TPR) befindet sich am C-Terminus; eine Transmembrandomäne (TM) ist am N-Terminus lokalisiert; eine Coiled-coil-Domäne (linker) liegt dazwischen. Die TPR-Domäne von FLU Domäne interagiert mit dem C-terminalen Ende der Glutamyl-tRNA Reduktase (GluTR), dem geschwindigkeitsbestimmenden Enzym der ALA-Synthese. Diese Arbeit zur Erweiterung des Wissen über die Funktion von FLU im Licht sowie über die Rolle der funktionellen Domänen von FLU bei der Inaktivierung der ALA-Synthese bei.
Lignocellulosic biomass is an abundant and valuable raw material for high commercial value and great industrial importance for bio-based chemical production, including levulinic acid as a relevant product. Levulinic acid (LA) is a versatile and specialty chemical in the green chemistry field with relevant applications in varied domains. Traditionally, the most widely used processes for producing LA are the homogeneous and heterogeneous acid hydrolysis of carbohydrates. There are few reports on the biological-based production of LA, so it remains a challenge. The development of metabolic engineering pathways for levulinic acid is definitively in the early stage and, at the moment, cannot meet the requirements for industrial production. Therefore, there is a need to bridge the gap between the well-developed chemical conversion processes and the underdeveloped biological conversion processes for levulinic acid.
Tetrapyrroles play fundamental roles in crucial processes including photosynthesis, respiration, and catalysis. In plants, 5-aminolevulinic acid (ALA) is the common precursor of tetrapyrroles. ALA is synthesized from activated glutamate by the enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAAT). ALA synthesis is recognized as the rate-limiting step in this pathway. We aimed to explore the contribution of GSAAT to the control of ALA synthesis and the formation of a protein complex with GluTR. In Arabidopsis thaliana, two genes encode GSAAT isoforms: GSA1 and GSA2. A comparison of two GSA knockout mutants with the wild type revealed the correlation of reduced GSAAT activity and ALA-synthesizing capacity in leaves with lower chlorophyll content. Growth and green pigmentation were more severely impaired in gsa2 than in gsa1, indicating the predominant role of GSAAT2 in ALA synthesis. Interestingly, GluTR accumulated to higher levels in gsa2 than in the wild type and was mainly associated with the plastid membrane. We propose that the GSAAT content modulates the amount of soluble GluTR available for ALA synthesis. Several different biochemical approaches revealed the GSAAT-GluTR interaction through the assistance of GluTR-binding protein (GBP). A modelled structure of the tripartite protein complex indicated that GBP mediates the stable association of GluTR and GSAAT for adequate ALA synthesis.
The C5 pathway in bacteria is responsible for the synthesis of 5-aminolevulinic acid, which forms the core skeleton of cofactors required for metabolism. One of the key actors in this pathway is a pyridoxamine-5'-phosphate (PMP)/pyridoxal-5'-phosphate (PLP) dependent enzyme called glutamate-1-semialdehyde aminomutase (GSAM). In this study, we crystallized the expression product of the uncharacterized pa4088 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The resulting high-resolution structure confirms it to be a member of the GSAM family. Continuous electron density indicates the presence of a PLP cofactor with a Schiff base linkage between the PLP cofactor and the ε-amino group of Lys286. A crystal structure of a K286A mutant in complex with PMP is also reported. As GSAM enzymes are not present in mammalian cells, this work provides a starting point for the investigation of GSAM as a target for drug development against P. aeruginosa infection.
