Directed Evolution of P 450 BM 3 into a p‐Xylene Hydroxylase

Regioselective hydroxylation of aromatic rings is a “dream reaction” in organic chemistry and is of high interest for the production of precursors for pharmaceuticals and flavors. In particular, the demand for phenol and alkylphenols as a feedstock for resins, plastics, and bisphenol A has increased constantly throughout the last century. Phenols are produced typically through the three-step cumene process, also known as the “Hock Process”. Alternatively, they can be isolated from natural sources such as coal tar, biomass, or by gasification processes. The latter often require intensive extraction procedures with high energy consumption, especially for the separation of isomeric alkylphenols. The “Hock Process” is highly profitable, in spite of its low product yields ( 5 % of the initially used benzene is converted to phenol) ; this owes mainly to the valuable acetone byproduct. Novel synthetic routes to phenols focus mainly on the development of metal catalysts, which generate reactive oxygen species for the hydroxylation of benzene educts. Direct hydroxylation with enzymes can be an attractive alternative; it reduces reaction and purification steps, as well as waste generation and process energy demands. Protein engineering methods such as semi-rational design and directed evolution allow these biocatalysts to be tailored to cost-effective production conditions. Cytochrome P 450 monooxygenases (CYPs), a protein superfamily that contains more than 5000 cloned members, catalyze oxygenation reactions with molecular oxygen at room temperature. Direct aromatic ring hydroxylation is a synthetically attractive reaction that has been reported for several P 450 monooxygenases. Whitehouse et al. recently described the rational driven design of the P 450 monooxygenase BM 3 (CYP 102 A 1) towards regioselective aromatic hydroxylation of substituted benzenes. A reaction mechanism for understanding aromatic hydroxylation of oand m-xylene has been proposed, however p-xylene was not investigated as a substrate. The p-xylene isomer was reported to be exclusively hydroxylated at the a-position. 10] Only a few enzymes (toluene 4-monooxygenases and microsomal P 450 monooxygenases) were reported to catalyze the aromatic hydroxylation of p-xylene to the corresponding phenol. 12] Unfortunately, enzymes such as toluene 4-monooxygenase suffer from low activity (kcat = 36 min ), stability (four-protein complex), and selectivity (20 % aromatic and 80 % benzylic hydroxylation) with p-xylene as the substrate. Nonetheless, direct aromatic hydroxylation of p-xylene to 2,5-dimethylphenol (2,5-DMP) is of high interest for the production of temperature stable polymers (>500 K), as is 2,5-DMP itself, which can act as a building block for “next-generation” plastics. We describe the semi-rational engineering of the self-sufficient monooxygenase P 450 BM 3 towards the direct aromatic hydroxylation of p-xylene. To achieve this goal, we selected and iteratively saturated five amino acid residues—Arg 47 (R 47), Tyr 51 (Y 51), Phe 87 (F 87), Ala 330 (A 330), and Ile 401 (I 401)—that had been reported to influence either substrate selectivity, activity, or both (Figure 2). 9, 14, 15] Besides using a sensitive and regioselective screening assay for phenols, we confirmed all products from P 450 BM 3 hydroxylation by GC (see Figure 1 and Experimental Section). Unexpectedly, the wild-type P 450 BM 3 showed a low activity for p-xylene (kcat = 68 min ; 45 % coupling efficiency), producing 2,5-DMP with high selectivity (>98 %), which contradicts a previous report. The latter finding can be attributed to higher substrate concentrations (1–30 mm p-xylene) used in the reaction setup. The recently proposed mechanism for oand m-xylene hydroxylation, however, does not exclude aromatic hydroxylation of p-xylene. In a first round of P 450 BM 3 mutagenesis, positions R 47/Y 51 were saturated simultaneously because both are key residues in controlling substrate access to the binding pocket (Figure 2). We obtained a variant M 1 (R 47 S/Y 51 W) with a more than seven-fold increase in product formation (kcat = 500 min 1 2,5-DMP) compared with the wild-type P 450 BM 3 (Table 1). Besides the increase in activity, variant M 1 showed a remarkable catalytic performance: the coupling efficiency increased by 20 % (total coupling 54 %). This was accompanied by a slightly decreased KM (from 7.9 to 7.1 mm). The bulky tryptophan residue on position 51 is thought to promote aromat-