General Features of P450cam and P450BM3

P450cam catalyzes the stereospecific oxidation of camphor to 5-exo-hydroxycam-phor (Scheme 4.1). It was the first P450 enzyme to be structurally characterized [26, 27], identifying the amino acid side-chains that define the active site and contact the substrate. Camphor is bound by numerous non-covalent contacts and a hydrogen bond between the camphor carbonyl and the phenol side-chain of Y96, leading to a specific orientation with C5 directly above the heme iron (Fig. 4.1). Atkins and Sligar were the first to investigate the effects of active site

Fig. 4.1 The active structure of P450cam with bound camphor showing the residues that contact the substrate. The hydrogen bond between the camphor carbonyl and Y96 side-chain plays a crucial role in orientating the substrate for regiospecific oxidation at C5 which is located above the heme. Hydrogen binding between the proximal thiolate and amide NH of L358 modulates the heme redox potential.

Fig. 4.1 The active structure of P450cam with bound camphor showing the residues that contact the substrate. The hydrogen bond between the camphor carbonyl and Y96 side-chain plays a crucial role in orientating the substrate for regiospecific oxidation at C5 which is located above the heme. Hydrogen binding between the proximal thiolate and amide NH of L358 modulates the heme redox potential.

changes on P450cam activity and substrate specificity by introducing the Y96F substitution to remove the hydrogen bond to camphor [28]. This mutation weakened camphor binding by a factor of 2, and other oxidation products (total 8%) were observed in addition to 5-exo-hydroxycamphor. Hence the main role of the hydrogen bond is not tight camphor binding but to orientate the substrate within the binding site and determine the regioselectivity of oxidation.

The role of hydrophobic enzyme/substrate contacts was explored with norcam-phor and 1-methylnorcamphor. For wild-type P450cam, the absence of methyl substituents in norcamphor compared to camphor lowered the selectivity to 45% of the 5-exo-hydroxy product, but the presence of just one methyl group in 1-methylnorcamphor raised the selectivity to 82% [29]. The V295I mutation increased the selectivity for the 5-exo product for norcamphor oxidation; the larger Ile side-chain compensated for the two methyl groups missing in 1-methylnorc-amphor compared with camphor. This was the first example of using active site mutations in P450 enzymes to compensate for structural differences between a target compound and the natural substrate.

Montellano and co-workers modeled the binding of non-natural substrates by wild-type P450cam and the L244A mutant using a computer docking protocol. When the turnover rates of these substrates were subsequently determined they showed significant correlation with the modeling data [30, 31]. Nicotine binding and oxidation by wild-type P450cam has been investigated [32]. Computer modeling agreed with the product being from oxidation at the 5' methylene group. However, the crystal structure showed nicotine N-coordination to the heme Fem center in an unproductive binding orientation. The structure of the FeII(CO) complex, which could serve as a model for the FeII(O2) complex in the catalytic cycle, showed that the nicotine nitrogen was displaced from the heme iron and the resultant binding orientation was consistent with 5' methylene oxidation [33]. This redox-induced switch might also operate in other P450 enzymes for substrates with a hetero-atom with an available lone pair of electrons.

P450BM-3 (CYP102A1) is a fusion protein composed of the N-terminal P450 monooxygenase domain linked to the C-terminal diflavin reductase domain. This protein organization is responsible for the high activity of this enzyme compared with other known P450s. Recently other members of the CYP102A family have been characterized (e.g. CYP102A2 and CYP102A3 from Bacillus subtilis) [34]. All three enzymes catalyze hydroxylation of medium to long-chain saturated fatty acids (Scheme 4.2) as well as epoxidation of unsaturated fatty acids. The physiological role of these fatty acid hydroxylases in Bacillus strains is unclear, but their high activity and selectivity towards iso- and anteiso-fatty acids suggest a role in regulating membrane fluidity [35]. P450BM-3 and its mutant A74G/F87V/L188Q demonstrated activity (up to 1200 min-1) towards a range of highly branched fatty acids and gave only one product in each reaction [36]. Highly branched fatty acids have polyketide-like structures. Selective functionalization of such substances could offer convenient routes to biologically active compounds such as macrolide antibiotics.

Scheme 4.2

Myristic acid

Scheme 4.2

High-resolution X-ray crystal structures are available for substrate-free [37], palmitoleic acid-bound [38], and N-palmitoylglycine-bound [39] forms of P450BM-3. Ligand binding has been studied by solution and solid-state NMR methods [40, 41]. The active site of P450BM-3 (Fig. 4.2) consists of a long hydrophobic channel, extending from the heme to the protein surface [38]. Analysis of crystal structures, followed by site-directed mutagenesis, revealed the important role of R47 and Y51 at the entrance of the substrate access channel. These two residues interact with the carboxylate moiety and are thus crucial for the proper positioning of fatty acid substrates. The guanidinium group of R47 plays a particularly prominent role [42, 43], while substitutions at Y51 appeared to have less impact [44]. The R47E, R47A, and R47G mutants retained their activity towards C12-C16 fatty

Fig. 4.2 The active site structure of P450BM-3 with palmitoleic acid (Palm) bound. The substrate carboxylate group interacts with R47 and Y51 at the entrance of the substrate channel while the aliphatic chain winds its way towards the heme. The F87 side-chain blocks access of the substrate to the heme iron and must move out of the way during the catalytic cycle to facilitate substrate oxidation.

Fig. 4.2 The active site structure of P450BM-3 with palmitoleic acid (Palm) bound. The substrate carboxylate group interacts with R47 and Y51 at the entrance of the substrate channel while the aliphatic chain winds its way towards the heme. The F87 side-chain blocks access of the substrate to the heme iron and must move out of the way during the catalytic cycle to facilitate substrate oxidation.

acids, but the kcat/Km values were 5- to 15-fold lower than those of the wild type [43-45].

The highly conserved active site residue F87 is important for correct orientation of the fatty acid hydrocarbon chain. Comparison of substrate-free and substrate-bound crystal structures of P450BM-3 revealed a substantial conformational difference that is caused by the F87 phenyl ring [37, 38]. Mutations at F87 can affect the activity and selectivity [46]. An unfavorable substitution F87 could lead to irreversible conformational changes during catalytic turnover which resulted in a decrease or complete loss of catalytic competence [44].

Beating The Butt On Your Own

Beating The Butt On Your Own

Need To Stop Smoking? Are You Willing To Follow My Powerful Strategies To Stop Smoking And Vividly Transform Your Life Today? Proven Tips, Tools and Tactics To Stop Smoking And Live An Awesome Life You Always Wanted.

Get My Free Ebook


Post a comment