Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Structural-Functional Correlations between Unique N-terminal Region and C-terminal Conserved Motif in Short-chain cis-Prenyltransferase from Tomato.

Neryl diphosphate (C10) synthase (NDPS1), a homodimeric soluble cis-prenyltransferase from tomato, contains four disulfide bonds, including two inter-subunit S-S bonds in the N-terminal region. Mutagenesis studies demonstrated that the S-S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C-terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well-ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C-terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site-directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N-terminal regions and the closed/open states of C-terminal regions may cooperate and be important for the catalytic mechanism of NDPS1.

Structural insights into a bacterial ß-glucosidase capable of degrading sesaminol triglucoside to produce sesaminol: toward the understanding of the aglycone recognition mechanism by the C-terminal lid domain.

The sesaminol triglucoside (STG)-hydrolyzing ß-glucosidase from Paenibacillus sp. (PSTG1), which belongs to glycoside hydrolase family 3 (GH3), is a promising catalyst for the industrial production of sesaminol. We determined the X-ray crystal structure of PSTG1 with bound glycerol molecule in the putative active site. PSTG1 monomer contained typical three domains of GH3 with the active site in domain 1 (TIM barrel). In addition, PSTG1 contained an additional domain (domain 4) at the C-terminus that interacts with the active site of the other protomer as a lid in the dimer unit. Interestingly, the interface of domain 4 and the active site forms a hydrophobic cavity probably for recognizing the hydrophobic aglycone moiety of substrate. The short flexible loop region of TIM barrel was found to be approaching the interface of domain 4 and the active site. We found that n-heptyl-ß-D-thioglucopyranoside detergent acts as an inhibitor for PSTG1. Thus, we propose that the recognition of hydrophobic aglycone moiety is important for PSTG1-catalyzed reactions. Domain 4 might be a potential target for elucidating the aglycone recognition mechanism of PSTG1 as well as for engineering PSTG1 to create a further excellent enzyme to degrade STG more efficiently to produce sesaminol.