RESUMEN
Chlorogenic acids (CHLs) are known to competitively bind to translocase-I (T1) of the glucose-6-phosphatase (G6 Pase) system, thereby inhibiting the transport of glucose-6-phosphate (G6P). This competitive binding results in a consequential reduction in blood sugar levels. In this study, steered molecular dynamics (SMD) simulation is employed to investigate the interaction between T1 and G6P, aiming to gain insights into the binding dynamics and diffusion process of G6P through T1. A database comprising 41 CHLs sourced from various plants was developed, subjected to minimization, and screened against T1 through conventional docking methods. The docked conformations were fed into a newly developed customized scoring method incorporating contact-based weights to assess the binding affinities that systematically rank and identify the most effective competitive inhibitors. Among the screened CHLs, 1-methoxy 3,5-dicaffeoylquinic acid, 3,4 dicaffeoyl quinic acid, and 3,4,5-tricaffeoylquinic acid stood out as the top three inhibitors, showcasing crucial atomic interactions with key residues within the binding pocket of T1, and these CHLs are sourced from readily available plants, diminishing reliance on coffee as the predominant CHL source. Along with the devised scoring function, which serves as a valuable tool for virtual screening and lead optimization in drug development, this study also marks a pioneering effort as it involves the modeling of the human translocase and unravels the mechanism of binding and diffusion of G6P within human T1, providing valuable insights into the structural prerequisites for successfully inhibiting the G6P system, laying the foundation for a rational approach to drug design. This research contributes to the progress of drug discovery strategies focused on the G6P system, presenting potential therapeutic avenues for addressing metabolic disorders linked to an impaired glucose metabolism.
RESUMEN
Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.
