Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development. For this purpose, two chimeric esterases AAM7 and PAR, constructed v...Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development. For this purpose, two chimeric esterases AAM7 and PAR, constructed via domain swapping between two ancient thermophilic esterases, were investigated on their molecular simulation（including homology modeling, substrates docking and substrate binding affinity validation） and enzymatic assay（specific activities and activation energies calculating）. Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated, the substrate binding domains or binding pockets are important but not the only factor for substrate preference.展开更多
Rubisco and fructose-1,6-bisphosphate aldolases （FBAs） are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. G...Rubisco and fructose-1,6-bisphosphate aldolases （FBAs） are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sati- vum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former spe- cies. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro- Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.展开更多
The origination of new genes is important for generating genetic novelties for adaptive evolution and biological diversity.However, their potential roles in embryonic development, evolutionary processes into ancient n...The origination of new genes is important for generating genetic novelties for adaptive evolution and biological diversity.However, their potential roles in embryonic development, evolutionary processes into ancient networks, and contributions to adaptive evolution remain poorly investigated. Here, we identified a novel chimeric gene family, the chiron family, and explored its genetic basis and functional evolution underlying the adaptive evolution of Danioninae fishes. The ancestral chiron gene originated through retroposition of nampt in Danioninae 48–54 million years ago(Mya) and expanded into five duplicates(chiron1–5) in zebrafish 1–4 Mya. The chiron genes(chirons) likely originated in embryonic development and gradually extended their expression in the testis. Functional experiments showed that chirons were essential for zebrafish embryo development. By integrating into the NAD^(+) synthesis pathway, chirons could directly catalyze the NAD^(+) rate-limiting reaction and probably impact two energy metabolism genes(nmnat 1 and naprt) to be under positive selection in Danioninae fishes. Together,these results mainly demonstrated that the origin of new chimeric chiron genes may be involved in adaptive evolution by integrating and impacting the NAD^(+) biosynthetic pathway. This coevolution may contribute to the physiological adaptation of Danioninae fishes to widespread and varied biomes in Southeast Asian.展开更多
基金Supported by the National Basic Research Program of China（Nos.2012CB721000, 2011CBA00800） and the National Natural Science Foundation of China（No.30970632）.
文摘Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development. For this purpose, two chimeric esterases AAM7 and PAR, constructed via domain swapping between two ancient thermophilic esterases, were investigated on their molecular simulation（including homology modeling, substrates docking and substrate binding affinity validation） and enzymatic assay（specific activities and activation energies calculating）. Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated, the substrate binding domains or binding pockets are important but not the only factor for substrate preference.
文摘Rubisco and fructose-1,6-bisphosphate aldolases （FBAs） are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sati- vum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former spe- cies. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro- Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.
基金This project is supported by the Pilot projects(XDB13020100)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB31000000)the National Natural Science Foundation of China(NSFC,31601859).
文摘The origination of new genes is important for generating genetic novelties for adaptive evolution and biological diversity.However, their potential roles in embryonic development, evolutionary processes into ancient networks, and contributions to adaptive evolution remain poorly investigated. Here, we identified a novel chimeric gene family, the chiron family, and explored its genetic basis and functional evolution underlying the adaptive evolution of Danioninae fishes. The ancestral chiron gene originated through retroposition of nampt in Danioninae 48–54 million years ago(Mya) and expanded into five duplicates(chiron1–5) in zebrafish 1–4 Mya. The chiron genes(chirons) likely originated in embryonic development and gradually extended their expression in the testis. Functional experiments showed that chirons were essential for zebrafish embryo development. By integrating into the NAD^(+) synthesis pathway, chirons could directly catalyze the NAD^(+) rate-limiting reaction and probably impact two energy metabolism genes(nmnat 1 and naprt) to be under positive selection in Danioninae fishes. Together,these results mainly demonstrated that the origin of new chimeric chiron genes may be involved in adaptive evolution by integrating and impacting the NAD^(+) biosynthetic pathway. This coevolution may contribute to the physiological adaptation of Danioninae fishes to widespread and varied biomes in Southeast Asian.
