Abstract
BackgroundBiosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.ResultsRecombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).ConclusionsThe successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.
Highlights
Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with flavin adenine dinucleotide (FAD) as a cofactor
Enzyme characteristics of KsdD3 KsdD3 is a FAD-dependent enzyme that catalyzes the formation of a double bond at the C1–2 position of 3-ketosteroid substrates (Fig. 1a)
KsdD3 lost almost all the activity at 45 °C. 3-ketosteroid-∆1-dehydrogenase catalyzes the reaction of substrates in organic solvents and detergents using hydrophobic interactions because of the negligible solubility of steroidal substrates in buffers
Summary
Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. Mao et al Microb Cell Fact (2018) 17:141 cleavage because of its high tolerance to organic solvents and remarkable bioconversion [14,15,16], involving the ∆1-dehydrogenation of steroids This dehydrogenation is a crucial modification in steroid synthesis because it increases the biological activity and economical value of the original steroidal substrate [17, 18]. The crystal Structure of KstD1 from Rhodococcus erythropolis revealed that Tyr487 and Gly491 promoted tautomerization, and Tyr318/Tyr119 and FAD abstracted a proton and a hydride ion, respectively [33, 34] These researches provided a fundamental understanding of KstD family enzyme, and the theoretical support for rational design of A. simplex KsdD3
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