The chalcone synthase superfamily of type III polyketide synthases.

FeII/alpha-ketoglutarate (alphaKG)-dependent hydroxylases catalyze an amazing diversity of reactions that result in protein side-chain modifications, repair of alkylated DNA/RNA, biosynthesis of antibiotics and plant products, metabolism related to lipids, and biodegradation of a variety of compounds. These enzymes possess a beta-strand "jellyroll" structural fold that contains three metal-binding ligands found in a His1-X-Asp/Glu-Xn-His2 motif. The cosubstrate, alphaKG, chelates FeII using its C-2 keto group (binding opposite the Asp/Glu residue) and C-1 carboxylate (coordinating opposite either His1 or His2). Oxidative decomposition of alphaKG forms CO2 plus succinate and leads to the generation of an FeIV-oxo or other activated oxygen species that hydroxylate the primary substrate. The reactive oxygen species displays alternate reactivity in related enzymes that catalyze desaturations, ring expansions, or ring closures. Other enzymes resemble the FeII/alphaKG-dependent hydroxylases in terms of protein structure or chemical mechanism but do not utilize alphaKG as a substrate. This review describes the reactions catalyzed by this superfamily of enzymes, highlights key active site features revealed by structural studies, and summarizes results from spectroscopic and other approaches that provide insights into the chemical mechanisms.

FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes.

Recent advances in the biosynthesis and accumulation of anthocyanins.

Salicin-based phenolic glycosides, hydroxycinnamate derivatives and flavonoid-derived condensed tannins comprise up to one-third of Populus leaf dry mass. Genes regulating the abundance and chemical diversity of these substances have not been comprehensively analysed in tree species exhibiting this metabolically demanding level of phenolic metabolism. Here, shikimate-phenylpropanoid pathway genes thought to give rise to these phenolic products were annotated from the Populus genome, their expression assessed by semiquantitative or quantitative reverse transcription polymerase chain reaction (PCR), and metabolic evidence for function presented. Unlike Arabidopsis, Populus leaves accumulate an array of hydroxycinnamoyl-quinate esters, which is consistent with broadened function of the expanded hydroxycinnamoyl-CoA transferase gene family. Greater flavonoid pathway diversity is also represented, and flavonoid gene families are larger. Consistent with expanded pathway function, most of these genes were upregulated during wound-stimulated condensed tannin synthesis in leaves. The suite of Populus genes regulating phenylpropanoid product accumulation should have important application in managing phenolic carbon pools in relation to climate change and global carbon cycling.

Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus.

Structure and function of the chalcone synthase superfamily of plant type III polyketide synthases

Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation.

The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl-CoAs with N-methylanthraniloyl-CoA or 4-coumaroyl-CoA to produce acridone alkaloids and flavonoid pigments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chalcone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding regions of these three cDNAs differ only marginally, and the translated polypeptides show about 90% identity with the CHSs from Citrus sinensis but less than 75% with the Ruta endogeneous ACSs. CHS1 was functionally expressed in Eschericha coli and its substrate specificity compared with those of the recombinant ACS1 and ACS2. 4-Coumaroyl-CoA was the preferred starter substrate for CHS1, but cinnamoyl-CoA and caffeoyl-CoA were also turned over at significant rates. However, N-methylanthraniloylCoA was not accepted. In contrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4-coumaroyl-CoA, which on average reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined with N-methylanthraniloyl-CoA as the starter substrate, while the conversion of cinnamoyl-CoA was negligible with both ACSs. The condensation mechanism of the acridone ring system differs from that of chalcone/flavanone formation. Nevertheless, our results suggest that very minor changes in the sequences of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site-directed mutagenesis.

Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens

Flavanone 3beta-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding. The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3beta-hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208-210 nm and a maximum at 191-193 nm which are characteristic for alpha-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding.

Site‐directed mutagenesis of the active site serine290 in flavanone 3β‐hydroxylase from Petunia hybrida

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2800%2901116-9

Inga Gröning

Papers

Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation.

Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens

Site‐directed mutagenesis of the active site serine290 in flavanone 3β‐hydroxylase from Petunia hybrida

The monomeric polypeptide comprises the functional flavanone 3β-hydroxylase from Petunia hybrida