Crystal Structure of P450cin in a Complex with Its Substrate, 1,8-Cineole, a Close Structural Homologue to d-Camphor, the Substrate for P450cam^,

Abstract

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to d-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and d-camphor are C(10) monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 A resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.

Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

“…Contrary to the P450s mentioned above, in the structure of MTCYP51 (ligand free [1h5z] versus inhibitor bound (4-phenylimidazole [1e9x], fluconazole [1ea1]) or the estriol (substrate analog) bound form [1×8v]) the BC and FG loop regions are superimposed very well, with the exception of the C-terminal part of the BC-loop, which is not seen in the ligand-free and estriol-bound MTCYP51; the active site volume remaining the largest amongst known P450 structures, 2,600 Å 3 [14]. For comparison, the calculated active site volumes of some other CYPs are: 256 and 264 Å 3 for cytochrome P450cin (CYP176A) and cytochrome P450cam (CYP101) [15]), 645 Å 3 for CYP2C5 [16], 670Å 3 for CYP2C9 [17], 1440 Å 3 for CYP2C8 [18] and 520 to 1386 Å 3 for CYP3A4 [19 and 20, respectively]. An assumption that the distant location of the F/G segment (~30 Å from the heme iron) may be largely preserved in the substrate-bound sterol 14α-demethylase suggested that substrate recognition sites (SRS) 2 and 3 [21] in this CYP family (or perhaps particularly in MTCYP51) might be non-functional [22].…”

Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

“…It is significant in this context that P450 cin is very closely related to the prototypical enzyme, P450 cam , not only in structure but also with respect to the size, shape, and chemical composition of cineol and camphor, their respective substrates [11]. Although the crystal structure of P450 cin complexed with cineol has been determined, it does not fully explain how P450 cin compensates for the missing Thr hydroxyl group.…”

“…Although the crystal structure of P450 cin complexed with cineol has been determined, it does not fully explain how P450 cin compensates for the missing Thr hydroxyl group. As noted in the introduction, several roles can be envisioned for the conserved distal Thr [11]. The finding that replacement of the hydroxyl of Thr252 with a methoxy group in P450 cam yields an active, coupled enzyme indicates that the role of the hydroxyl is likely to be as a proton acceptor rather than proton donor [7].…”

“…The crystal structure of CYP176A suggested that Asn242, the corresponding residue in the I-helix, forms a hydrogen bond with 1,8-cineole [11]. It has also been suggested that this residue, which is part of a stable hydrogen bonding network, substitutes for the conserved Thr and helps to deliver the protons required for oxygen activation [11]. Asn242 may therefore fulfill a dual function by both anchoring the substrate and assisting in proton delivery [11].…”

“…It has also been suggested that this residue, which is part of a stable hydrogen bonding network, substitutes for the conserved Thr and helps to deliver the protons required for oxygen activation [11]. Asn242 may therefore fulfill a dual function by both anchoring the substrate and assisting in proton delivery [11]. In this investigation, we have examined whether the role of Asn in P450 cin is specific to that enzyme or is a substitution that is more generally effective in maintaining the P450 catalytic function.…”