Diversified amino acid-mediated allosteric regulation of phosphoglycerate dehydrogenase for serine biosynthesis in land plants.

The phosphorylated pathway of serine biosynthesis is initiated with 3-phosphoglycerate dehydrogenase (PGDH). The liverwort Marchantia polymorpha possesses an amino acid-sensitive MpPGDH which is inhibited by l-serine and activated by five proteinogenic amino acids, while the eudicot Arabidopsis thaliana has amino acid-sensitive AtPGDH1 and AtPGDH3 as well as amino acid-insensitive AtPGDH2. In this study, we analyzed PGDH isozymes of the representative land plants: the monocot Oryza sativa (OsPGDH1-3), basal angiosperm Amborella trichopoda (AmtriPGDH1-2), and moss Physcomitrium (Physcomitrella) patens (PpPGDH1-4). We demonstrated that OsPGDH1, AmtriPGDH1, PpPGDH1, and PpPGDH3 were amino acid-sensitive, whereas OsPGDH2, OsPGDH3, AmtriPGDH2, PpPGDH2, and PpPGDH4 were either sensitive to only some of the six effector amino acids or insensitive to all effectors. This indicates that PGDH sensitivity to effectors has been diversified among isozymes and that the land plant species examined, except for M. polymorpha, possess different isozyme types in terms of regulation. Phylogenetic analysis suggested that the different sensitivities convergently evolved in the bryophyte and angiosperm lineages. Site-directed mutagenesis of AtPGDH1 revealed that Asp538 and Asn556 residues in the ACT domain are involved in allosteric regulation by the effectors. These findings provide insight into the evolution of PGDH isozymes, highlighting the functional diversification of allosteric regulation in land plants.

Identification and Biochemical Characterization of the Serine Biosynthetic Enzyme 3-Phosphoglycerate Dehydrogenase in Marchantia polymorpha.

L-serine is an important molecule in all living organisms, and thus its biosynthesis is considered to be regulated according to demand. 3-Phosphoglycerate dehydrogenase (PGDH), the first committed enzyme of the phosphorylated pathway of L-serine biosynthesis, is regulated by negative feedback from L-serine in bacteria. In the case of the vascular plant Arabidopsis thaliana, two PGDH isozymes out of three are inhibited by L-serine and activated by L-alanine, L-valine, L-methionine, L-homoserine, and L-homocysteine, suggesting a more complicated regulatory mechanism of L-serine biosynthesis in A. thaliana than in bacteria. However, it remains to be clarified whether the activation mechanism of PGDH by amino acids is conserved in land plants. In this study, we identified the sole isozyme of PGDH in the liverwort Marchantia polymorpha (MpPGDH) and elucidated its biochemical characteristics. MpPGDH cDNA encodes a 65.6 kDa protein that contains a putative transit peptide for chloroplast localization. MpPGDH shares 75-80% identity with A. thaliana isozymes and forms a homotetramer in vitro. Recombinant MpPGDH exhibited an optimal pH of 9.0, apparent Michaelis constants of 0.49 ± 0.04 and 0.096 ± 0.010 mM for 3-PGA and NAD+, respectively, and apparent maximum velocity of 5.65 ± 0.10 µmol⋅min-1⋅mg-1, similar to those of A. thaliana isozymes. Phosphate ions were found to stabilize MpPGDH, suggesting that phosphate ions are also a crucial factor in the regulation of serine biosynthesis via the phosphorylated pathway in Marchantia polymorpha. MpPGDH was inhibited by L-serine in a cooperative manner and was activated by L-alanine, L-valine, L-methionine, L-homoserine, and L-homocysteine to a lesser extent than it is in A. thaliana. The results suggest that an ancestral PGDH of land plants was inhibited byL-serine and slightly activated by five other amino acids.

Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana.

The proteinogenic amino acid L-serine is a precursor for various essential biomolecules in all organisms. 3-Phosphoglycerate dehydrogenase (PGDH) is the first committed enzyme of the phosphorylated pathway of L-serine biosynthesis, and is regulated by negative feedback from L-serine in bacteria and plants. In the present study, two Arabidopsis PGDH isoforms were inhibited by L-serine but were activated by L-amino acids such as L-homocysteine in vitro. Activation and inhibition by these amino acids was cooperative, suggesting an allosteric mechanism. Moreover, the half maximal effective concentration of L-homocysteine was 2 orders of magnitude lower than that of L-serine, suggesting greater regulatory potency. These are the first data to show that PGDH is activated by various biomolecules and indicate that serine biosynthesis is regulated by multiple pathways.

Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway.

Acetoacetyl-CoA is the precursor of 3-hydroxy-3-methylglutaryl (HMG)-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. Acetoacetyl-CoA is also the precursor of poly-beta-hydroxybutyrate, a polymer belonging to the polyester class produced by microorganisms. The de novo synthesis of acetoacetyl-CoA is usually catalyzed by acetoacetyl-CoA thiolase via a thioester-dependent Claisen condensation reaction between two molecules of acetyl-CoA. Here, we report that nphT7, found in the mevalonate pathway gene cluster from a soil-isolated Streptomyces sp. strain, encodes an unusual acetoacetyl-CoA synthesizing enzyme. The recombinant enzyme overexpressed in Escherichia coli catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-(acyl carrier protein) resulted in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity was detected through the condensation of two molecules of acetyl-CoA. Based on these properties of NphT7, we propose to name this unusual enzyme of the thiolase superfamily acetoacetyl-CoA synthase. Coexpression of nphT7 with the HMG-CoA synthase gene and the HMG-CoA reductase gene in a heterologous host allowed 3.5-fold higher production of mevalonate than when only the HMG-CoA synthase and HMG-CoA reductase genes were expressed. This result suggests that nphT7 can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leading to the production of useful terpenoids and poly-beta-hydroxybutyrate.