Marco Bocola

Iterative Saturation Mutagenesis Accelerates Laboratory Evolution of Enzyme Stereoselectivity: Rigorous Comparison with Traditional Methods

Efficacy in laboratory evolution of enzymes is currently a pressing issue, making comparative studies of different methods and strategies mandatory. Recent reports indicate that iterative saturation mutagenesis (ISM) provides a means to accelerate directed evolution of stereoselectivity and thermostability, but statistically meaningful comparisons with other methods have not been documented to date. In the present study, the efficacy of ISM has been rigorously tested by applying it to the previously most systematically studied enzyme in directed evolution, the lipase from Pseudomonas aeruginosa as a catalyst in the stereoselective hydrolytic kinetic resolution of a chiral ester. Upon screening only 10,000 transformants, unprecedented enantioselectivity was achieved (E = 594). ISM proves to be considerably more efficient than all previous systematic efforts utilizing error-prone polymerase chain reaction at different mutation rates, saturation mutagenesis at hot spots, and/or DNA shuffling, pronounced positive epistatic effects being the underlying reason.

Directed Evolution of an Enantioselective Epoxide Hydrolase: Uncovering the Source of Enantioselectivity at Each Evolutionary Stage

Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments, and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.

Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity

An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2‐methyl‐decanoic acid p‐nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E=1.1 for the wild‐type to E=51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular‐dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).

Learning from directed evolution: Theoretical investigations into cooperative mutations in lipase enantioselectivity

Molecular modeling with classical force‐fields has been used to study the reactant complex and the tetrahedral intermediate in lipase‐catalyzed ester hydrolysis in 20 enzyme/substrate combinations. The R and S enantiomers of α‐methyldecanoic acid ester served as substrates for the wild‐type lipase from Pseudomonas aeruginosa and nine selected mutants. After suitable preparation of initial structures from an available wild‐type crystal structure, each system was subjected to 1 ns CHARMM force‐field molecular dynamics simulations. The resulting geometric and energetic changes allow interpretation of some experimentally observed effects of mutations, particularly with regard to the “hot spots” at residues 155 and 162. The replacement S155F enhances S enantiopreference through a steric relay involving Leu162. The double mutation S53P + L162G improves S enantioselectivity by creating a new binding pocket for the S enantiomer with an additional stabilizing hydrogen bond to His83. The simulations provide insight into remote and cooperative effects of mutations.

A Conclusive Mechanism of the Photoinduced Reaction Cascade in Blue Light Using Flavin Photoreceptors

On the basis of extensive first-principle calculations within the framework of quantum mechanics/molecular mechanics (QM/MM), a conclusive mechanism for the formation of the signaling state of blue light using flavin (BLUF) domain proteins is proposed which is compatible with the experimental data presently available. Time-dependent density functional, as well as advanced coupled cluster response theory was employed for the QM part in order to describe the relevant excited states. One of the key residues involved in the mechanism is the glutamine adjacent to the flavin chromophore. The reaction cascade, triggered by the initial photoexcitation of the flavin chromophore, involves isomerization of this residue but no rotation as assumed previously. The fact that only the environment, but not the flavin chromophore by itself, is chemically transformed along the individual steps of the mechanism is unique for biological photoreceptors. The final isomer of the glutamine tautomer, i.e., the imidic acid, is further stabilized by the interchange of a methionine residue in the binding pocket with a tryptophan residue. The flip of these two residues might be the trigger for the large conformational change of this protein which is consequently transmitted as the signal to the biological environment.

Theoretical study on the repair mechanism of the (6-4) photolesion by the (6-4) photolyase.

UV irradiation of DNA can lead to the formation of mutagenic (6-4) pyrimidine-pyrimidone photolesions. The (6-4) photolyases are the enzymes responsible for the photoinduced repair of such lesions. On the basis of the recently published crystal structure of the (6-4) photolyase bound to DNA [Maul et al. 2008] and employing quantum mechanics/molecular mechanics techniques, a repair mechanism is proposed, which involves two photoexcitations. The flavin chromophore, initially being in its reduced anionic form, is photoexcited and donates an electron to the (6-4) form of the photolesion. The photolesion is then protonated by the neighboring histidine residue and forms a radical intermediate. The latter undergoes a series of energy stabilizing hydrogen-bonding rearrangements before the electron back transfer to the flavin semiquinone. The resulting structure corresponds to the oxetane intermediate, long thought to be formed upon DNA-enzyme binding. A second photoexcitation of the flavin promotes another electron transfer to the oxetane. Proton donation from the same histidine residue allows for the splitting of the four-membered ring, hence opening an efficient pathway to the final repaired form. The repair of the lesion by a single photoexcitation was shown not to be feasible.

Revisiting the Lipase from Pseudomonas aeruginosa: Directed Evolution of Substrate Acceptance and Enantioselectivity Using Iterative Saturation Mutagenesis

The most thoroughly studied enzyme in directed evolution is the lipase from Pseudomonas aeruginosa (PAL) as a catalyst in the hydrolytic kinetic resolution of 2-methyldecanoic acid p-nitrophenyl ester. Seminal studies utilized epPCR, saturation mutagenesis and DNA shuffling or combinations thereof. With current emphasis on efficacy in laboratory evolution, however, we recently applied our previously developed method, iterative saturation mutagenesis (ISM), to the same catalytic system, discovering that this approach is much more efficient than the original strategies. Herein, we consider PAL once more, this time testing ISM as a means to broaden the substrate scope of this lipase by studying bulky substrates of the type 2-phenylalkanoic acid esters as substrates that are not accepted by the WT. Highly active and enantioselective (E up to 436) mutants were evolved, a process that required only small mutant libraries and thus a minimum of screening effort. A theoretical investigation using molecular dynamics simulations and docking experiments revealed the source of enhanced activity and stereoselectivity.

A single amino acid substitution converts benzophenone synthase into phenylpyrone synthase

Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of BPS was rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. 3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor substrate for PPS. The aryl moiety of the triketide intermediate may be trapped in the new pocket by hydrogen bond formation with the backbone, thereby acting as an inhibitor. PPS is a promising biotechnological tool for manipulating benzoate-primed biosynthetic pathways to produce novel compounds.

Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis

Xanthones are natural products present in plants and microorganisms. In plants, their biosynthesis starts with regioselective cyclization of 2,3′,4,6-tetrahydroxybenzophenone to either 1,3,5- or 1,3,7-trihydroxyxanthones, catalysed by cytochrome P450 (CYP) enzymes. Here we isolate and express CYP81AA-coding sequences from Hypericum calycinum and H. perforatum in yeast. Microsomes catalyse two consecutive reactions, that is, 3′-hydroxylation of 2,4,6-trihydroxybenzophenone and C–O phenol coupling of the resulting 2,3′,4,6-tetrahydroxybenzophenone. Relative to the inserted 3′-hydroxyl, the orthologues Hc/HpCYP81AA1 cyclize via the para position to form 1,3,7-trihydroxyxanthone, whereas the paralogue HpCYP81AA2 directs cyclization to the ortho position, yielding the isomeric 1,3,5-trihydroxyxanthone. Homology modelling and reciprocal mutagenesis reveal the impact of S375, L378 and A483 on controlling the regioselectivity of HpCYP81AA2, which is converted into HpCYP81AA1 by sextuple mutation. However, the reciprocal mutations in HpCYP81AA1 barely affect its regiospecificity. Product docking rationalizes the alternative C–O phenol coupling reactions. Our results help understand the machinery of bifunctional CYPs.

Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis

A number of model structures of the CalA suggested by comparative modeling were tested by site‐directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.