Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates

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. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0981-0) contains supplementary material, which is available to authorized users.

Universal Capability of 3-Ketosteroid Δ1-Dehydrogenases to Catalyze Δ1-Dehydrogenation of C17- Substituted Steroids

“…erythropolis WY 1406 strain can also convert substrates with extended C17 substituent, like 21-acetoxy-pregna-4,9(11),16-triene-3,20-dione. In addition to AcmB and the new AcmB2 [8,20], there were some literature reports about KSTDs activity towards steroids with a more complex side chain on the C17 position [17][18][19]22,47]. Zhang et al described five KSTD isoenzymes from Gordonia neofelifaecis of which four exhibited activity toward cholest-4-en-3-one.…”

“…Although KSTDs substrate scope is generally broad, most of the enzymes prefer steroids with small substituents on the C17 position [2]. Only a few of them exhibited activity towards more branched substrates, such as cholest-4-en-3-one, 3-oxo-5β-cholan-24-oic acid, the methyl ester of 3-oxo-4-cholen-24-oic acid, cortisone acetate, hydrocortisone acetate or 21-acetoxy-pregna-4,9(11),16-triene-3,20-dinone [17][18][19][20][21][22]. The comparative study of substrate specificity is additionally hindered by poor or very bad solubility of steroids in the water phase in which all kinetic enzyme assays are conducted [8].…”

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

“…Although KSTDs substrate scope is generally broad, most of the enzymes prefer steroids with small substituents on the C17 position [ 2 ]. Only a few of them exhibited activity towards more branched substrates, such as cholest-4-en-3-one, 3-oxo-5β-cholan-24-oic acid, the methyl ester of 3-oxo-4-cholen-24-oic acid, cortisone acetate, hydrocortisone acetate or 21-acetoxy-pregna-4,9(11),16-triene-3,20-dinone [ 18 – 23 ]. The comparative study of substrate specificity is additionally hindered by poor or very bad solubility of steroids in the water phase in which all kinetic enzyme assays are conducted [ 8 ].…”

Expression, Purification, Refolding and Characterization of A Neverland Protein from Caenorhabditis Elegans

“…In order to purify the CeNVD_ pET-28a(+), the washed cells were resuspended in 30 mL lysis buffer A (20 mM Tris-HCl, 20 mM imidazole, 500 mM NaCl, and 1 mM dithiothreitol, pH 8.0) containing 0.5 mg/mL lysozyme and 1 mM phenylmethane sulfonyl uoride (PMSF) and disrupted using a sonicator (Sonic Dismembrator Model 100, Pittsburgh, PA) on ice bath for 20 min, the unbroken cells and cell debris were removed by centrifugation at 20,000 ×g for 30 min at 4 °C, the supernatant was applied to a nickelnitrilotriacetic acid (Ni-NTA) agarose a nity chromatography matrix (Qiagen, Hilden, Germany), preequilibrated with lysis buffer A, after washing the open column with 10 mL lysis buffer A extensively, the bound protein was eluted with 10 mL elution buffer A (20 mM Tris-HCl, 300 mM imidazole, 300 mM NaCl, and 1 mM dithiothreitol, pH 8.0) [22].…”