Protein Engineering of the Progesterone Hydroxylating P450‐Monooxygenase CYP17A1 Alters Its Regioselectivity

Abstract

The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17-C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active-site residues were identified and systematically mutated to alanine. In whole-cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α-hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.

Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

“…However, studies with the Ala370Ile or Ala370Leu variants gave a regioselectivity reversal and produced predominantly C 5 ‐hydroxylation instead . Similarly, with triple or quintuple mutants of P450 3A4, the activation of progesterone gave dominant 2β‐hydroxylation rather than 6β‐hydroxylation and, hence, affected substrate positioning and activation …”

Rational and semi-rational engineering of cytochrome P450s for biotechnological applications

“…For example, the CYP17A1 is known to catalyze the 17α-hydroxylation of pregnenolone and progesterone as well as the subsequent C17-C20 bond cleavage, providing important precursors for different pathways [ 32 , 33 ]. To study the reaction of progesterone with bovine CYP17A1, a homology model was first created based on the crystal structures of human CYP17A1 (PDB codes: 4NKW , 4NKY , and 5IRQ ), Danio rerio CYP17A1 (PDB code: 4R1Z ) and CYP17A2 (PDB code: 4R20 ), and was then used a guide for alanine scanning of conserved active-site residues by site-directed mutagenesis, revealing that L206A, V366A, and V483A mutations alter regioselectivity, increasing the formation of the side-product, 16α-hydroxyprogesterone, up to 40% of the total product formation [ 34 ].…”

Regioselectivity of Cobalamin‐Dependent Methyltransferase Can Be Tuned by Reaction Conditions and Substrate

“…Regioselectivity may by altered or tuned by various options like by enzyme engineering [18] or substrate and medium engineering. [17,19] To learn about the influence of the reaction medium and the substituents on the regioselectivity of the MTase I for methylation of 4-substituted catechols 1 b-i (Scheme 2), three parameters were investigated: (i) the use of co-solvents, (ii) the nature of the substituent in position 4 and (iii) the pH.…”