Curse and Blessing of Non-Proteinogenic Parts in Computational Enzyme Engineering.

Enzyme engineering aims to improve or install a new function in biocatalysts for applications ranging from chemical synthesis to biomedicine. For decades, computational techniques have been developed to predict the effect of protein changes and design new enzymes. However, these techniques may have been optimized to deal with proteins composed of the standard amino acid alphabet, while the function of many enzymes relies on non-proteogenic parts like cofactors, nucleic acids, and post-translational modifications. Enzyme systems containing such molecules might be handled or modeled improperly by computational tools, and thus be unsuitable, or require additional tweaking, parameterization, or preparation. In this review, we give an overview of common and recent tools and workflows available to computational enzyme engineers. We highlight the various pitfalls that come with including non-proteogenic compounds in computations and outline potential ways to address common issues. Finally, we showcase successful examples from the literature that computationally engineered such enzymes.

Stabilization of cyclohexanone monooxygenase by computational and experimental library design.

Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature-sensitive enzymes catalyzing the Baeyer-Villiger reaction, which display excellent stereo- and regioselectivity and offer a green alternative to the commonly used, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI-31, a substrate-promiscuous enzyme that efficiently catalyzes the production of the nylon-6 precursor ε-caprolactone. We used a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein-stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13°C and an approximately 33x increase in half-life at 30°C. In contrast to the wild-type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications.

Overriding Traditional Electronic Effects in Biocatalytic Baeyer-Villiger Reactions by Directed Evolution.

Controlling the regioselectivity of Baeyer-Villiger (BV) reactions remains an ongoing issue in organic chemistry, be it by synthetic catalysts or enzymes of the type Baeyer-Villiger monooxygenases (BVMOs). Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates, which are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4-phenyl-2-butanone as substrate, the regioselectivity was reversed from 99:1 (wild-type enzyme in favor of the normal product undergoing 2-phenylethyl migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective reaction using the synthetic reagent m-CPBA, which provides solely the normal product. Reversal of regioselectivity was also achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity in reactions of 4-phenyl-2-butanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein engineering of BVMOs.

Exploring the Substrate Scope of Baeyer-Villiger Monooxygenases with Branched Lactones as Entry towards Polyesters.

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that are able to convert cyclic ketones into lactones by the insertion of oxygen. The aim of this study was to explore the substrate scope of several BVMOs with (biobased) cyclic ketones as precursors for the synthesis of branched polyesters. The product structure and the degree of conversion of several biotransformations were determined after conversions by using self-sufficient BVMOs. Full regioselectivity towards the normal lactones of jasmatone and menthone was observed, whereas the oxidation of other substrates such as α,ß-thujone and 3,3,5-trimethylcyclohexanone resulted in mixtures of regioisomers. This exploration of the substrate scope of both established and newly discovered BVMOs towards biobased ketones contributes to the development of branched polyesters from renewable resources.

Polycyclic Ketone Monooxygenase from the Thermophilic Fungus Thermothelomyces thermophila: A Structurally Distinct Biocatalyst for Bulky Substrates.

Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.

Manipulating the stereoselectivity of the thermostable Baeyer-Villiger monooxygenase TmCHMO by directed evolution.

Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S). In order to induce the reversal of enantioselectivity of this reaction as well as the transformations of other substrates, directed evolution based on iterative saturation mutagenesis (ISM) was applied, leading to (R)-selectivity (94% ee) without affecting the thermostability of the biocatalyst.

Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs.

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.

The Expanded Central Dogma: Genome Resynthesis, Orthogonal Biosystems, Synthetic Genetics.

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction [i.e., (re)synthesis] rather than deconstruction (analysis). In this sense, biological sciences now follow the lead given by the chemical sciences. Synthesis can complement analytic studies but also allows novel approaches to answering fundamental biological questions and opens up vast opportunities for the exploitation of biological processes to provide solutions for global problems. In this review, we explore aspects of this synthesis paradigm as applied to the chemistry and function of nucleic acids in biological systems and beyond, specifically, in genome resynthesis, synthetic genetics (i.e., the expansion of the genetic alphabet, of the genetic code, and of the chemical make-up of genetic systems), and the elaboration of orthogonal biosystems and components.

A Computational Library Design Protocol for Rapid Improvement of Protein Stability: FRESCO.

The ability to stabilize enzymes and other proteins has wide-ranging applications. Most protocols for enhancing enzyme stability require multiple rounds of high-throughput screening of mutant libraries and provide only modest improvements of stability. Here, we describe a computational library design protocol that can increase enzyme stability by 20-35 °C with little experimental screening, typically fewer than 200 variants. This protocol, termed FRESCO, scans the entire protein structure to identify stabilizing disulfide bonds and point mutations, explores their effect by molecular dynamics simulations, and provides mutant libraries with variants that have a good chance (>10%) to exhibit enhanced stability. After experimental verification, the most effective mutations are combined to produce highly robust enzymes.

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

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.