Switching Cofactor Dependence of 7β-Hydroxysteroid Dehydrogenase for Cost-Effective Production of Ursodeoxycholic Acid

Abstract

Dehydrogenases are widely employed as biocatalysts for the production of optically pure chemicals under mild conditions. Most dehydrogenases are nicotinamide cofactor (NADPH or NADH)-dependent oxidoreductases. 7β-Hydroxysteroid dehydrogenase (7β-HSDH) is a key enzyme for the biochemical synthesis of ursodeoxycholic acid (UDCA). To date, all reported 7β-HSDHs are strictly NADPH-dependent enzymes. However, compared with NADPH, NADH is much more economical, making it the preferential cofactor for synthetic applications of dehydrogenases. In this work, a recombinant 7β-HSDH originating from Ruminococcus torques was rationally engineered to alter its cofactor dependence using a strategy referred to as Cofactor Specificity Reversal: Small-and-Smart Library Design (CSR-SaSLiD), which is based on structural information and conservative sequence alignment. We rationally designed a small-and-smart library containing only five mutants that enabled the quick identification of target variants. Compared with the wild type, the resultant mutant, G39D, showed a 953 000-fold switch in cofactor specificity from NADPH to NADH, and another mutant, G39D/T17A, resulted in 223-fold enhanced activity with NADH. The structural mechanism regarding the effect of mutation on the reversal of cofactor preference and improvement of catalytic activity was elucidated with the aid of molecular dynamics simulation. Furthermore, it was confirmed that the CSR-SaSLiD strategy can be extended to other 7β-HSDHs. This work provides an efficient approach to altering cofactor preference and subsequently recovering the enzymatic activity of dehydrogenases for cost-effective biotechnical applications.

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

“…Thanks to these interesting features, HSDHs have been widely studied during the last years for their exploitation in the biocatalyzed synthesis of key intermediates of the drug ursodeoxycholic acid (UDCA), a largely applied therapeutic agent used for the dissolution of cholesterol gallstones and for the treatment of different hepatic diseases. These efforts have led to the characterization of different enzymes showing either 7α‐, 7β‐ or 12α‐HSDH activity, as well as to the development of biocatalyzed processes for the preparation of UDCA intermediates …”

“…These efforts have led to the characterization of different enzymes showing either 7α-, 7β-or 12α-HSDH activity, [2,3] as well as to the development of biocatalyzed processes for the preparation of UDCA intermediates. [8][9][10][11] The ability of enzymes to act on substrates possessing chemical structures significantly different from their natural ones is a quite common feature. [12][13][14] This "substrate promiscuity" was also observed with HSDHs a long time ago by Davies and coworkers who reported the activity of a 3α,20β-HSDH from Mortierella ramanniana toward 3-hydroxybicyclo-heptan-6ones.…”

Application of Ketoreductase in Asymmetric Synthesis of Pharmaceuticals and Bioactive Molecules: An Update (2018–2020)

“…[25] Compared with chemical synthesis, this biosynthesis approach is more efficient and environmentally friendly. To make this process more economic‐feasible, their most recent work was rationally engineering the cofactor preference of a 7β‐HSDH from Ruminococcus torques (7β‐ Rt HSDH) from NADPH to NADH by using a strategy termed Cofactor Specificity Reversal: Small‐and‐Smart Library Design (CSR‐SaSLiD), which was based on structural information and conservative sequence alignment [26] . G39D/T17 A, the best mutant discovered, exhibited 223‐fold enhanced activity with NADH compared to the wild‐type enzyme.…”

Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome

“…Enzymatic synthesis of dipeptides has become a popular research topic because of its excellent selectivity and environment-friendly manner [ 2 , 3 ]. Carnosine has been considered to be synthesized by the called carnosine synthase from β-alanine and histidine in many tissues.…”