Crystal Structure of CYP106A2 in Substrate‐Free and Substrate‐Bound Form

Abstract

CYP106A2 from Bacillus megaterium ATCC 13368 is known as a bacterial steroid hydroxylase that is also capable of hydroxylating a variety of terpenoids. To analyze the substrate specificity of this enzyme further, different resin acids of the abietane and pimarane types were tested with regard to binding and conversion. Product formation could be shown for all tested substrates. Spectroscopic studies revealed type I binding spectra for isopimaric acid, but dehydroabietic acid did not induce a high-spin shift of the enzyme. Interestingly, binding of abietic acid resulted in a type II difference spectrum typical for nitrogenous inhibitors. Co-crystallization of CYP106A2 with abietic acid and structure determination revealed bending of the heme cofactor when abietic acid was bound in the active site. Quantum chemical calculations strongly suggest that this heme distortion is the cause of the unusual spectroscopic characteristics.

Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding

“…Both contain iron, which can undergo a change in its formal oxidation state. Although the Fe 2 S 2 cofactor of the FeS cluster is ligated by four cysteine residues and is located near to the surface of the ferredoxins, the heme group is more buried within CYP106A2 as seen in the X‐ray structure (pdb entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5IKI) (Fig. ).…”

“…The adrenodoxin‐like electron transfer protein 1 [Etp1(516–618)] was prepared likewise (pdb entry code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2WLB, chain B) . As the receptor, the conformation of CYP106A2 from B. megaterium in substrate bound form was used (pdb entry code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5IKI, chain B) . For protein–protein docking the ClusPro2 web service (https://cluspro.org/login.php) was used, applying default parameters .…”

“…Terpenoids make CYP106A2 very attractive for applications in biotechnology . The availability of the high‐resolution crystallographic structure of CYP106A2 does not only allow the application of molecular docking studies to assist the interpretation of product spectra but, furthermore, enables the rational design of mutants that show altered product distributions . Likewise, the choice of the redox partner – the ferredoxin, which delivers the electrons – influences the amount and kind of products being formed.…”

Effect of sildenafil on human aromatase activity: From in vitro structural analysis to catalysis and inhibition in cells

“…In a second simulation, the pentacoordinated heme cofactor was replaced by the water-bound hexacoordinated one from the crystal structure of CYP106A2 (PDB ID 4YT3) [23] and docking was again performed.…”

Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns

“…After identifying an efficient redox system for a specific P450, further optimizations by methods of protein engineering should be performed and finally tests with other P450 isoforms are designated. As a first step to create a universal redox system for P450s, we tested the applicability of diverse redox partners for the well-studied CYP106A2 6,15,40–42 . The experiments showed clearly that all used combinations of redox partners were able to provide electrons to CYP106A2, but only the truncated forms of Adx and Etp1, combined with AdR or Arh1, were able to support CYP106A2 in a way that almost all provided progesterone (200 µM) was converted within 30 min.…”