Chalcone synthase

CHS exists as a homodimeric protein with each monomer approximately 42-45 kDa in size.[6] Each monomer possesses a β-keto synthase (KS) activity that catalyzes the sequential head to tail incorporation of two-carbon acetate units into a growing polyketide chain. CHS contains a five layer αβαβα core, a location of the active site and dimerization interface that is highly similar to thiolase-fold containing enzymes. The dimerization interface contains both hydrophobic and hydrophilic residues and is generally flat except for a pair of N-terminal helices that lay entwined across the top. Although the helices are not involved in reaction, they may contain intracellular localization signals as in yeast thiolase. They may also undergo a conformational change to participate in the formation of transient multi-protein complexes with other enzymes in the various pathways diverging from the general phenylpropanoid biosynthetic pathway.

The enzyme is localized in the cytosol, associating with the endoplasmic reticulum membrane.[7] In another study, it was shown that CHS and CHI co-localize at the nucleus as well.[8]

There are two distinct bi-lobed active site cavities located at the bottom edge of each monomer’s αβαβα core. Identical six-residue loops, which meet at the dimer interface, separate the two active sites from each other. The loops being with Thr132 in the active site and ends with a cis-peptide bond to Pro138. A Met137 residue plugs a hole in the other monomer’s active site. Therefore, the active site is buried except for a 16 Å CoA-binding tunnel that connects the catalytic surface to the outer surrounding milieu. The width of the tunnel is too narrow for the aromatic substrates and products that must pass through it, implying that there must be some dynamic mobility within and around the tunnel when placed in solution.

The active site contains a conserved catalytic triad of Cys164, His303 and Asn336. These residues aid in multiple decarboxylation and condensation reactions, with Cys164 acting as the active site nucleophile. Phe215 and Phe265 are two other important amino acids that act as “gatekeepers” to block the lower protein of the opening between the CoA-binding tunnel and the active site cavity. This limits the access of water to the active site while accommodating substrates and intermediates of varying shapes and sizes. Phe215 also orients the substrates at the active site during elongation of the polyketide intermediate.

CHS is noncompetitively inhibited by flavanoid pathway products such as naringenin and chalcone naringenin.[10] Despite lack of direct evidence in vivo, flavonoids are believed to accumulate in the cytosol to a level that blocks CHS activity to avoid toxic levels in plants.[11]

CHS is constitutively expressed in plants but can also be subject to induced expression through light/ UV light and well as in response to pathogens, elicitors and wounding. The CHS promoter contains a G-box motif with a sequence of CACGTG. This has been shown to play a role in response to light.[12] Other light sensitive domains include Box I, Box II, Box III, Box IV or three copies of H-box (CCTACC).[9]

The chalcone synthase gene of Petunia plants is famous for being the first gene in which the phenomenon of RNA interference was observed; researchers intending to upregulate the production of pigments in light pink or violet flowers introduced a transgene for chalcone synthase, expecting that both the native gene and the transgene would express the enzyme and result in a more deeply colored flower phenotype. Instead the transgenic plants had mottled white flowers, indicating that the introduction of the transgene had downregulated or silenced chalcone synthase expression.[13] Further investigation of the phenomenon indicated that the downregulation was due to post-transcriptional inhibition of the chalcone synthase gene expression via an increased rate of messenger RNA degradation.[14]