Structural Analysis of CYP101C1 from Novosphingobium aromaticivorans DSM12444

Abstract

CYP101C1 from Novosphingobium aromaticivorans DSM12444 is a homologue of CYP101D1 and CYP101D2 enzymes from the same bacterium and CYP101A1 from Pseudomonas putida. CYP101C1 does not bind camphor but is capable of binding and hydroxylating ionone derivatives including α- and β-ionone and β-damascone. The activity of CYP101C1 was highest with β-damascone (k(cat)=86 s(-1)) but α-ionone oxidation was the most regioselective (98 % at C3). The crystal structures of hexane-2,5-diol- and β-ionone-bound CYP101C1 have been solved; both have open conformations and the hexanediol-bound form has a clear access channel from the heme to the bulk solvent. The entrance of this channel is blocked when β-ionone binds to the enzyme. The heme moiety of CYP101C1 is in a significantly different environment compared to the other structurally characterised CYP101 enzymes. The likely ferredoxin binding site on the proximal face of CYP101C1 has a different topology but a similar overall positive charge compared to CYP101D1 and CYP101D2, all of which accept electrons from the ArR/Arx class I electron transfer system.

Solution Structural Ensembles of Substrate-Free Cytochrome P450cam

“…The presence of an ordered water molecule hydrogen-bonded to the amide of Ile 88 in 3L62 indicates that this region is more solvent-accessible in 3L62 than in other CYP101A1 structures. We note that recently the crystal structure of an open form of the related CYP101C1 has been determined in which the X-Pro bond preceding the intact B’ helix is found to be in the trans conformation (32). Comparison of 3L62 with camphor-bound structure 3L63 also shows quite large rmsd (> 8 Å) displacements, although instead of the B-B’ loop and the β3-β4 sheet being the primary site of displacement, as we observe, the displacements in the crystallographic structures are localized in the F and G helices, the F-G loop and the “sprung” B’ helix (22).…”

X‐ray crystal structure of cytochrome P450 monooxygenase CYP101J2 fromSphingobium yanoikuyaestrain B2

“…In contrast, the entire region was resolved in the camphor bound structure . CYP101C1, despite being a member of CYP101, does not bind or oxidize camphor at all but hydroxylates ionone derivatives . This enzyme was crystallized with two different substrate‐like molecules bound and both showed an open conformation with the B' helix resolved.…”

“…The vast diversity of function is suggested to occur as a result of sequence and structural differences in the F and G helices and BC loop, which define the substrate‐binding channel . Crystal structures of multiple members of the family crystallized in both the presence and absence of substrate show that the substrate‐binding channel can exist both open and closed forms, and suggest a substrate‐dependent, multistep, substrate‐binding mechanism that is reliant on intrinsic dynamics of the access channel …”

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

“…With CYP101C1 from Novosphingobium aromaticivorans DSM12444, a homologue of CYP101A1 has been described very recently that does not bind camphor but is capable of binding and hydroxylating ionone derivatives including α-and β-ionone and β-damascone [54]. Scheme 1 gives an overview of P450 catalyzed ionone conversions.…”