Substrate Recognition and Molecular Mechanism of Fatty Acid Hydroxylation by Cytochrome P450 from Bacillus subtilis

Abstract

Cytochrome P450 isolated from Bacillus subtilis (P450 BS␤ ; molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the ␣-and ␤-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450 BS␤ in the substrate-bound form, determined at a resolution of 2.1 Å. P450 BS␤ exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg 242 guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the ␣-and ␤-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg 242 in the same fashion as has been reported for the active site of chloroperoxidase, His 105 -Glu 183 , which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450 BS␤ is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.Two bacterial cytochrome P450s isolated from Sphingomonas paucimobilis and Bacillus subtilis, P450 SP␣ 1 and P450 BS␤ , respectively, are heme-containing enzymes that catalyze the hydroxylation reaction of long chain fatty acids (e.g. myristic acid) using hydrogen peroxide (H 2 O 2 ) as an oxidant to produce hydroxylated (-OH) fatty acids (1, 2). In the enzymatic reactions, an oxygen atom derived from H 2 O 2 is efficiently introduced into the substrate with a high catalytic turnover (1,000 min Ϫ1 ) (2-4). P450 SP␣ produces the ␣-OH fatty acid (100%) as the product, whereas P450 BS␤ produces both the ␤-OH (60%) and the ␣-OH (40%) fatty acids (1, 2, 4, 5). The amino acid sequence of the two enzymes shares a 44% identity (2). Data base investigation has shown that P450 SP␣ and P450 BS␤ belong to the P450 superfamily and, therefore, they have been given the systematic nomenclature designations CYP152B1 and CYP152A1, respectively (6). However, when compared with reactions catalyzed by other P450s, two characteristic properties in the P450 SP␣ and P450 BS␤ reactions were found, i.e. the utilization of H 2 O 2 and the site specificity of the reaction.In typical P450 reactions an oxygen atom derived from molecular oxygen (O 2 ) is inserted into the substrates (7), and the reaction is referred to as a monooxygenation reaction. Two protons and two electrons are required in the monooxygenation reaction. The electrons are supplied from NAD(P)H through mediation by flavoproteins and iron-sulfur proteins, and the protons are probably delivered from solvent water to the active site through a specific hydrogen-bonding network (8). In the monooxygenase P450 system, H 2 O 2 is sometimes used as a surrogate for the O 2 /2e Ϫ /2H ϩ system (peroxide sh...

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

“…Extensive contact with hydrophobic aromatic amino acids (e.g., Phe291, Phe79) may serve to anchor EA and provide a source of radical stabilization. Although regiospecific Cβ−H atom abstraction would most certainly need to be primarily satisfied, it does not solely ensure subsequent overoxidation to furnish a hydrocarbon by OleT and related CYP152 orthologs (40,52). Interesting comparisons are found with the functional divergence observed for nonheme iron enzymes and synthetic complexes, which effectively negotiate • OH bond insertion, rebound of alternative radical species (22), and substrate desaturation (50,53).…”

“…We (35) and others (34) have postulated that alkene formation may proceed via initial substrate H atom abstraction based on the accepted mechanism for aliphatic hydroxylations by P450-I, loss of a hydrogen from the Cβ position during conversion of a C n fatty acid to a C n-1 alkene, and structural similarity of OleT (33) with fatty acid hydroxylases (40,42) that exhibit significantly large substrate 2 H steady-state KIEs (39). Appreciable accumulation of Ole-I was not previously detected in the OleT single-turnover reaction with protiated EA (H 39 -EA), suggesting that the rate of reaction was too fast to be captured in our earlier photodiode array (PDA) studies (35).…”

“…OleT differs from most P450s in two fundamental aspects. Belonging to the CYP152 family (39,40), catalysis is efficiently initiated by hydrogen peroxide, rather than O 2 and reducing equivalents delivered through a redox chain. Moreover, single (35) and multiple turnover (36,38) studies of what is generally believed to approximate the chain length of the physiological substrate, eicosanoic acid (EA), have indicated that oxidative decarboxylation, affording nonadecene, is the largely dominant reaction route (34).…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…Furthermore, binding of a carboxylate to the R242 was shown to be crucial for compound I formation. The stabilization of the aliphatic side chain of the substrate by a hydrophobic substrate channel was demonstrated [71]. To determine the importance of residues involved in the hydrophobic substrate channel, the F79L, V170F, and F79L/V170F variants were tested for hydroxylation of C14:0 (entries [18][19][20].…”

Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates

“…In these studies, P450 BSβ was chosen as the biocatalytic system because it is soluble, does not require a redox partner and can efficiently use hydrogen peroxide instead of oxygen and the expensive NADPH cofactor. Isolated from Bacillus subtilis, P450 BSβ (also known as P450 152A1) normally catalyzes the hydroxylation of long-chain fatty acid substrates such as myristic acid to form α-and β-hydroxymyristic acid [26].…”