Glycine N-acyltransferase (GLYAT; EC 2.3.1.13, Accession ID: AAI12537) is a key enzyme in mammalian homeostasis that has been linked to several pathologies in humans, including cancer. Here we report the first crystal structure of a member of the GLYAT family, both in the apo form as well as bound to benzoyl-CoA. Binding of glycine could be inferred from an acetate molecule from the crystallization solution. A detailed analysis of its structure and the effects of mutations of key residues helped elucidate the catalytic mechanism, showing a general base-catalyzed reaction driven by a potential low-barrier hydrogen bond (LBHB) formed between the catalytic Glu-His dyad. This work will aid further studies of GLYAT and other members of the family.

The rising global burden of infectious fungal disease and the steady increase of resistance by fungal pathogens to available treatments necessitate a concerted effort to develop new antifungal medicines. Squalene synthase is a critical enzyme in the synthesis of ergosterol, a fungal-specific sterol, and has not yet been exploited as an antifungal drug target for pathogenic yeast. In this study, we heterologously produce squalene synthase (SQS) from the opportunistic pathogen Candida albicans in Escherichia coli to obtain high-quality pure protein for structural studies. A library of nine C- or N&C-terminally truncated SQS variants was successfully produced and purified with immobilised metal-affinity chromatography (IMAC). Two variants were upscaled and purified to near-homogeneity, but size-exclusion chromatography showed possible aggregation. Nevertheless, this study provides a starting point to further optimise buffer conditions to produce high-quality heterologous SQS from C. albicans for downstream crystallography experiments. ### Competing Interest Statement The authors have declared no competing interest.

AbstractThe rising global burden of infectious fungal disease and the steady increase of resistance by fungal pathogens to available treatments necessitate a concerted effort to develop new antifungal medicines. Squalene synthase is a critical enzyme in the synthesis of ergosterol, a fungal-specific sterol, and has not yet been exploited as an antifungal drug target for pathogenic yeast. In this study, we heterologously produce squalene synthase (SQS) from the opportunistic pathogenCandida albicansinEscherichia colito obtain high-quality pure protein for structural studies. A library of nine C- or N&C-terminally truncated SQS variants was successfully produced and purified with immobilised metal-affinity chromatography (IMAC). Two variants were upscaled and purified to near-homogeneity, but size-exclusion chromatography showed possible aggregation. Nevertheless, this study provides a starting point to further optimise buffer conditions to produce high-quality heterologous SQS fromC. albicansfor downstream crystallography experiments.

Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) with peroxygenase activity are becoming the preferred biocatalysts for oxyfunctionalization reactions. While whole cells (WCs) or cell-free extracts (CFEs) of Escherichia coli are often preferred for cofactor-dependent monooxygenase reactions, hydrogen peroxide (H 2 O 2 ) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by E. coli are expected to quickly degrade H 2 O 2 . We used the CRISPR/Cas system to delete the catalase encoding chromosomal genes, katG , and katE , from E. coli BL21-Gold(DE3) to obtain a catalase-deficient strain. A short UPO, Dca UPO, and two CYP peroxygenases, Ssca CYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM H 2 O 2 was depleted within 10 min after addition to WCs and CFEs of the wild-type strain, at least 60% remained after 24 h in WCs and CFEs of the catalase-deficient strain. CYP peroxygenase reactions, with generally lower turnover frequencies, benefited the most from the use of the catalase-deficient strain. Comparison of purified peroxygenases in buffer versus CFEs of the catalase-deficient strain revealed that the peroxygenases in CFEs generally performed as well as the purified proteins. We also used WCs from catalase-deficient E. coli to screen three CYP peroxygenases, wild-type SscaCYP, Ssca CYP_E284A, and Ssca CYP_E284I for activity against 10 substrates comparing H 2 O 2 consumption with substrate consumption and product formation. Finally, the enzyme-substrate pair with highest activity, Ssca CYP_E284I, and trans -β-methylstyrene, were used in a preparative scale reaction with catalase-deficient WCs. Use of WCs or CFEs from catalase-deficient E. coli instead of purified enzymes can greatly benefit the high-throughput screening of enzyme or substrate libraries for peroxygenase activity, while they can also be used for preparative scale reactions.

The development of synthetic routes to produce enantiopure ( R )‐citronellal as a key intermediate for the synthesis of (–)‐menthol and other valuable terpenoids is highly relevant in the pharmaceutical, flavor, and fragrance industries. Herein, we showcase a cascade with two consecutive biocatalytic steps performed separately using the inherent selectivity of a short‐chain alcohol dehydrogenase (SDR) and an ene reductase (ERED) from the Old Yellow Enzyme (OYE) family. The first reaction involves the Aa SDR1‐catalyzed oxidation of relatively inexpensive geraniol in a biphasic system, providing geranial as an intermediate. The organic phase containing geranial is then extracted and transferred to the second step, where the ERED variant OYE2_Y83V catalyzes the asymmetric reduction of geranial to produce ( R )‐citronellal, achieving >90% conversion and >99% enantiomeric excess. The use of n ‐heptane in a two‐liquid phase system not only facilitates substrate and product solubilization but also minimizes geranial isomerization. This biocatalytic cascade therefore enables the synthesis of enantiopure ( R )‐citronellal.

Ene-reductases from the old yellow enzyme (OYE) family have been traditionally employed in the reduction of conjugated C & boxH;C double bonds. This study explores the underutilized oxidative potential of OYEs, demonstrating their capability to catalyze the enantioselective desaturation of carbonyl compounds. Utilizing a deprotonated tyrosine residue as a catalytic base, we developed a method to enable OYE-catalyzed desaturation at ambient temperature and alkaline pH without the need for high-temperature conditions. Through screening of various OYE enzymes, we identified several candidates from different genera with enhanced desaturase activity across different substrates. This work broadens the scope of biocatalytic applications for OYEs, introducing a novel approach to the synthesis of chiral alpha,beta-unsaturated carbonyl compounds. Old yellow enzymes can catalyze the desaturation of carbonyl compounds. In this reaction, the catalytic tyrosine needs to be deprotonated. Hence, at alkaline pH, desaturation is feasible at ambient temperature. In this report, we demonstrate the feasibility of this reaction, its generality and relevant factors influencing the reaction efficiency. image

Regioselective C-H functionalization of fatty acids and alcohols is a challenging reaction, especially in-chain/midchain hydroxylation. These hydroxy fatty acids or diols offer a synthetic route to valuable delta- and gamma-lactones. Although terminal and subterminal hydroxylation of fatty acids and alcohols by cytochrome P450 monooxygenases have been extensively explored, the molecular determinants of in-chain hydroxylation are unknown. Here we performed ancestral sequence reconstruction (ASR) of the subfamily of CYP505Es, able to perform in-chain hydroxylation, together with their closest related subterminal hydroxylases. Three ancestors were resurrected, which represented the in-chain and subterminal hydroxylases, as well as their common ancestor, which displayed little regioselectivity. Mutations were introduced to investigate the divergence in regioselectivity observed in the natural evolution. Whereas subterminal hydroxylation appears to be through multiple additive mutations in the active site, in-chain hydroxylation was greatly affected by the BC-loop. ASR provides not only insight for directed evolution studies but also more promiscuous ancestors as templates for the starting point for laboratory evolution.

Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C-C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with alpha,beta-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of alpha,beta-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten alpha,beta-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic alpha,beta-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R-selectivity of 83% ee. The best substrate was the aromatic alpha,beta-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R-selectivity >99.9% ee. Michaelis-Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover k(cat) of 0.71 +/- 0.03 s(-1) and a K-m of 2.46 +/- 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 & Aring; resolution was obtained, and docking studies showed a productive pose with the substrate.

Different strategies have previously been reported to convert cytochrome P450 monooxygenases to peroxygenases, allowing H2O2-driven oxyfunctionalization reactions. Comparison of the BM3 (CYP102A1) peroxygenase variant 21B3, obtained through enlargement of the active site by the F87A mutation followed by mutational stabilization towards H2O2, with the BM3_T268E variant, with an acid-base catalyst introduced, showed 21B3 to be the superior peroxygenase. A combination of these two strategies (21B3_T268E combinatorial mutant), however, resulted in reduced peroxygenase activity. The further introduction of the F(A)87V and A328F mutations (87–328 variants), previously reported to improve the regioselectivity of BM3 on n-alkanes, resulted in a loss of activity towards dodecanoic acid, a substrate commonly used to evaluate the activity of BM3. Although a reduction in activity was observed for styrene, the combinatorial mutants yielded higher enantioselectivities for R-styrene oxide. The activity towards α-olefins were, however, comparable between the different peroxygenase variants of BM3, but with absolute selectivity for epoxidation observed with the combinatorial mutants 21B3_87-328 and 21B3_T268E_87-382. Structural investigation of the active site architecture showed significant differences in the I-helix and heme accessibility. According to these results, future directed evolution studies will require selective pressure not only for H2O2, but also the specific substrate to be activated rather than surrogate substrates.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins with broad chemical functionality that have widespread application as powerful biocatalysts given their high stereo- and regio-selectivity. A large variety of value-added compounds, including pharmaceuticals and natural products, can be synthesized by P450s, often with superior selectivity and at milder conditions than their chemical counterparts making them ideal alternative catalysts. This review addresses the most recent advances in P450-mediated syntheses and include enzyme engineering strategies that have expanded the stereo- and regio-selectivity of P450 steroid and fatty acid hydroxylation as well as facilitated C-N bond formation and cyclization, all of which have synthetic applications to produce value-added compounds. Approaches to overcome the limitations of P450 catalysis will also be discussed, including the use of decoy molecules to expand the substrate scope and enable the use of hydrogen peroxide, self-sufficient systems, cascade reactions and cofactor alternatives to improve efficiency and biotechnological viability.

Cytochrome P450 monooxygenases (CYPs) are biocatalysts able to catalyze a variety of regio- and stereoselective oxyfunctionalization reactions using an iron(iv)-oxo porphyrin pi-cation radical prosthetic group, commonly referred to as compound I (Cpd I). The formation of Cpd I is, however, dependent on molecular oxygen and the sequential transfer of electrons from expensive nicotinamide cofactors via additional redox partner proteins. Recently, CYPs have been engineered to introduce or enhance peroxygenase activity, whereby Cpd I is formed from H2O2. Here we explore the potential of natural CYPs containing an aspartate instead of the conventional threonine on the distal side of the heme, for peroxygenase activity. Peroxygenase activity was demonstrated with three new CYPs, with SscaCYP from Streptomyces scabiei demonstrating the highest activity towards the hydroxylation of trans-beta-methyl styrene and the sulfoxidation of thioanisole. The X-ray crystal structure of SscaCYP revealed two potential acid-base catalysts (D241 and E284) in close proximity to the axial water molecule for the heterolytic O-O cleavage during H2O2 activation for Cpd I formation. Both side chain groups are also located at H2O channels to the bulk solvent. Mutagenesis of either side chain increased the peroxygenase activity, whereas removal of both abolished the peroxygenase activity of SscaCYP. Spectral analysis confirmed the tight binding of the axial water molecule when both carboxylate groups are present. With only one carboxylate group present lower concentrations of H2O2 are also required for catalysis, suggesting H2O and H2O2 exchange as a rate limiting factor in the wild-type SscaCYP. The improved peroxygenase activity and increased substrate scope of the SscaCYP variants, have identified two "hot-spots" for future protein engineering with one an alternative site for the introduction of an acid-base catalyst for engineering peroxygenase activity in other CYPs. Mild oxyfunctionalization reactions driven by hydrogen peroxide using cytochrome P450 monooxygenases with alternative heme environments.

γ- and δ-lactones are valuable flavor and fragrance compounds. Their synthesis depends on the availability of suitable hydroxy fatty acid precursors. Three short unspecific peroxygenases were identified that selectively hydroxylate the C4 and C5 positions of C8–C12 fatty acids to yield after lactonization the corresponding γ- and δ-lactones. A preference for C4 over C5 hydroxylation gave γ-lactones as the major products. Overoxidation of the hydroxy fatty acids was addressed via the reduction of the resulting oxo acids using an alcohol dehydrogenase in a bienzymatic cascade reaction.

Regioselective oxyfunctionalisation ofn-alkanes and production of non-vicinal diols by evolved CYP505A30 through rational transfer of knowledge between protein scaffolds.

We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (TsER) that combines control over facial selectivity in the reduction of electron deficient C??C double bonds with thermostability (up to 70 ?C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystallisation of TsER C25D/I67T and in silico docking. The TsER variant panel shows excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2R,5S-dihydrocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h-1 were achieved, which are comparable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also demonstrated the reusability of the reaction system by consecutively removing the organic phase (n-pentane) for product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L-1 R-levodione from a 45 mL aqueous reaction, containing less than 17 mg (10 ?M) enzyme and the reaction only stopping because of acidification. The TsER variant panel provides a robust, highly active and stereocomplementary base for further exploitation as a tool in preparative organic synthesis.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

The combination of heterogeneous catalysis and biocatalysis into one-pot reaction cascades is a potential approach to integrate enzymatic transformations into existing chemical infrastructure.

Regioselective aromatic hydroxylation is desirable for the production of valuable compounds. External flavin-containing monooxygenases activate and selectively incorporate an oxygen atom in phenolic compounds through flavin reduction by the nicotinamide adenine dinucleotide coenzyme, and subsequent reaction with molecular oxygen. This study provides the proof of principle of flavoenzyme-catalyzed selective aromatic hydroxylation with coenzyme biomimetics. The carbamoylmethyl-substituted biomimetic in particular affords full conversion in less than two hours for the selective hydroxylation of 5 mM 3- and 4-hydroxybenzoates, displaying similar rates as with NADH, achieving a 10 mM/h enzymatic conversion of the medicinal product gentisate. This biomimetic appears to generate less uncoupling of hydroxylation that typically leads to undesired hydrogen peroxide. Therefore, we show these flavoenzymes have the potential to be applied in combination with biomimetics.

Regioselective biocatalytic oxyfunctionalization of n-alkanes for the production of non-vicinal diols through sequential oxygenation by a cytochrome P450 monooxygenase.

Reductions play a key role in organic synthesis, producing chiral products with new functionalities. Enzymes can catalyse such reactions with exquisite stereo-, regio- and chemoselectivity, leading the way to alternative shorter classical synthetic routes towards not only high-added-value compounds but also bulk chemicals. In this review we describe the synthetic state-of-the-art and potential of enzymes that catalyse reductions, ranging from carbonyl, enone and aromatic reductions to reductive aminations.

Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called “uncoupling”, whereby NAD(P)H derived electrons are lost due to the reduced intermediates’ (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2–22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.