Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme

Abstract

Detoxifying enzymes such as flavin-containing monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question. Here, we addressed this issue by applying a cumulative alanine mutagenesis approach to cyclohexanone monooxygenase from Thermocrispum municipale, a flavin-dependent Baeyer-Villiger monooxygenase which we chose as a model system because of its pronounced thermostability and substrate promiscuity. Simultaneous removal of up to eight noncatalytic active-site side chains including four phenylalanines had no effect on protein folding, thermostability, and cofactor loading. We observed a linear decrease in activity, rather than a selectivity switch, and attributed this to a less efficient catalytic environment in the enlarged active-site space. Time-resolved kinetic studies confirmed this interpretation. We also determined the crystal structure of the enzyme in complex with a mimic of the reaction intermediate that shows an unaltered overall protein conformation. These findings led us to propose that this cyclohexanone monooxygenase may lack a distinct substrate selection mechanism altogether. We speculate that the main or exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates.

Scheme 1. Baeyer-Villiger Reaction and Catalytic Mechanism of BVMOs Showing the Various Oxidation States of the Flavin (Fl)

Figure 1

Overall structure of TmCHMO crystallized in complex with hexanoic acid (a, PDB code 6GQI) and superposition with a model of the peroxyflavin (b) and the Criegee intermediate (c). (a) Asymmetric unit of the TmCHMO crystal structure depicting the secondary structure (left monomer) and as surface representation (right monomer). The surface is cut open (gray planes) to emphasize the position of the ligand molecules in the tunnel and active site. The NADPH and FAD domains are colored red and pink, respectively. NADP⁺, FAD, and hexanoic acids are shown as ball and sticks colored green, yellow, and cyan, respectively. (b and c) The flavin and NADP⁺ cofactors are shown as ball and sticks with yellow and green carbons, respectively. In b, hexanoic acid as crystallized is shown as ball and sticks (cyan carbons) superimposed on a model of the peroxyflavin. In c, a model of the Criegee intermediate (Scheme 1) of hexanal is shown together with the electron density of the bound hexanoic acid (weighted 2F_o–F_c map, contoured at the σ = 1.2 level; Table S1).

Figure 2

Active-site pocket and tunnel of TmCHMO wild type (crystal structure, top panel) and the 8× alanine mutant (model, bottom panel). The left and right panels are the same scene rotated by 180°. FAD and NADP⁺ are depicted in yellow and green, respectively. All active-site residues are displayed as sticks in various colors. The surface they create and which forms the pocket is in the same color. In the mutant, residues that contribute to the newly shaped pocket are also shown with gray carbons. The hexanoic acid ligand bound in the tunnel is depicted as ball and sticks. The rest of the protein is shown as a gray surface representation, cut open at various planes (black). The active-site pocket is not cut, to emphasize the volume differences between wild type and mutant. As a result, the inner hexanoic acid ligand, bound close to the flavin (Figure 1b,c), cannot be seen.

Figure 3

A summary of the thermostability, kinetic, and bioconversion properties of the TmCHMO active-site mutants (see Table 1). The thermostability and kinetic properties of the mutants are shown in a and b. BCH is rac-bicyclo-[3.2.0]hept-2-en-6-one. (c) Conversions of individual substrates are quantitative and plotted on a logarithmic scale. The enzyme concentration was 2 μM. 2-Butanone conversions were performed using whole cells. More information can be found in the Supporting Information. (d) Conversions of substrate mixes are semiquantitative and approximated based on GC peaks as full (>99%), moderate (50–99%), low (5–50%), or trace (<5%). The enzyme concentration was 10 μM. Panel e illustrates product regioselectivity (abnormal vs normal lactone). (f) Legend for c–e. All data sets with an error bar (corresponding to ± sd) are from two, the remaining data from one independent experiment.

Figure 4

Tunnel mutagenesis strategy and activity results. (a) Surface representation of the substrate tunnel of TmCHMO occurring in the crystal structure. (b) Schematic cross-section through TmCHMO’s active site and substrate tunnel and contributing residues. All residues depicted in the same color and corresponding to ring-like dissections of the tunnel were simultaneously mutated to alanine. (c and d) Conversion results for substrate mixes (c, semiquantitative, see Figure 3) and of cyclohexanone and rac-bicyclo-[3.2.0]hept-2-en-6-one (rac-BCH) in individual conversions (d, quantitative). Enzyme concentrations were 2 μM and 10 μM for mixed and individual substrates, respectively. (e) Stability of the ring-wise tunnel multialanine mutants; the dashed line indicates the melting temperature of the wild-type enzyme.