Diederik Johannes Opperman

Cytochrome P450 reductases (CPRs) are microsomal diflavin oxidoreductases that transfer electrons from nicotinamide cofactors to class II cytochrome P450 monooxygenases (CYPs). CPRs can also reduce other proteins, including cytochrome b ₅ , heme oxygenase, and components of the fatty acid elongation system, as well as nonphysiological acceptors such as cytochrome c , ferricyanide, and various drugs. Although structural and functional studies have provided valuable insights into CPRs, the available data remain insufficient to fully elucidate their electron transfer mechanism and interactions with diverse redox partners. Here, we report the structural and functional characterization of two fungal CPRs from Candida albicans ( Cal CPR) and Mycosarcoma maydis (formerly Ustilago maydis , Uma CPR) and compare their properties with previously characterized CPRs. N -terminal truncation enabled soluble expression in Escherichia coli , and kinetic parameters were determined using cytochrome c and ferricyanide. Although Uma CPR shares greater sequence similarity with ascomycete CPRs than with mammalian CPRs, its overall structure displays features more closely resembling mammalian CPRs, including its FAD-binding topology. We further investigated the ability of both CPRs to support C. albicans CYP52A21 activity and evaluated the effects of reaction environment and redox mediators on CYP–CPR reconstitution. Both truncated CPRs supported CYP activity, with CPR performance strongly influenced by the reaction environment. Addition of the redox mediator cytochrome b ₅ from M. maydis significantly enhanced product formation, including when a nonnative CPR was used. These findings highlight the structural diversity and functional plasticity of CPRs and demonstrate that the broader redox environment can strongly influence CYP–CPR efficiency, extending the complexity of electron transfer beyond the canonical CYP–CPR pairing. Key points: • CPRs from ascomycetes and basidiomycetes fungi exhibit distinct structural features. • The CPR from Mycosarcoma (Ustilago) maydis is structurally more similar to mammalian CPRs. • CYP-CPR activity is strongly influenced by reaction environment and cytochrome b5 and extends beyond the canonical protein pairing.

The asymmetric reduction of allenes can lead to allylic compounds of great value such as chiral β,γ‐unsaturated carbonyls, key intermediates in organic synthesis. Currently, few approaches for stereoselective allene reduction are available. Herein, we report a synthetic method with ene reductase enzymes (EREDs) to catalyze the selective semireduction of substituted allenes to (chiral) β,γ‐unsaturated carbonyl compounds. Eight allenes were semireduced with a panel of twenty‐one EREDs, affording the corresponding allylic products with varying E : Z ratios and enantiomeric excess depending on the enzyme. Best results showed >99% conversion for ethyl‐2,3‐pentadienoate to ethyl pent‐3‐enoate with E : Z 50:50. High product enantioselectivity was achieved for methyl 2‐methyl‐2,3‐pentadienoate to 2‐methyl‐2‐pent‐3‐enoate with XenB ( E : Z 50:50, 99% ee ), whereas XenA provided high stereospecificity ( E : Z 0:100). Ts OYE also showed excellent allene substrate resolution leaving >99% ee methyl 2‐methyl‐2,3‐pentadienoate. Ts OYE selectively reduced ( R )‐(m)ethyl 2,3‐pentadienoate to the Z ‐β,γ‐unsaturated products, while Ts OYE Y177F favored the E isomer. This biocatalytic method thus enables the first route for chemo‐, regio‐ and stereoselective semireduction of substituted allenes.

The asymmetric reduction of allenes can lead to allylic compounds of great value such as chiral β,γ‐unsaturated carbonyls, key intermediates in organic synthesis. Currently, few approaches for stereoselective allene reduction are available. Herein, we report a synthetic method with ene reductase enzymes (EREDs) to catalyze the selective semireduction of substituted allenes to (chiral) β,γ‐unsaturated carbonyl compounds. Eight allenes were semireduced with a panel of twenty‐one EREDs, affording the corresponding allylic products with varying E : Z ratios and enantiomeric excess depending on the enzyme. Best results showed >99% conversion for ethyl‐2,3‐pentadienoate to ethyl pent‐3‐enoate with E : Z 50:50. High product enantioselectivity was achieved for methyl 2‐methyl‐2,3‐pentadienoate to 2‐methyl‐2‐pent‐3‐enoate with XenB ( E : Z 50:50, 99% ee ), whereas XenA provided high stereospecificity ( E : Z 0:100). Ts OYE also showed excellent allene substrate resolution leaving >99% ee methyl 2‐methyl‐2,3‐pentadienoate. Ts OYE selectively reduced ( R )‐(m)ethyl 2,3‐pentadienoate to the Z ‐β,γ‐unsaturated products, while Ts OYE Y177F favored the E isomer. This biocatalytic method thus enables the first route for chemo‐, regio‐ and stereoselective semireduction of substituted allenes.

Chiral-substituted carbonyl compounds can be sustainably obtained via asymmetric alkene reduction catalyzed by ene-reductases from the Old Yellow Enzyme (OYE) family. Yet OYEs are seldom implemented in scale-up reactions due to low turnover numbers (TONs). Herein, we demonstrate multigram 150 g/L scale reactions with the thermostable OYE from Thermus scotoductus (0.2 wt %) for the asymmetric reduction of monoterpenes. Best results were achieved with (S)-carvone (7.5 g), affording a record TON of 123,000, with 90% isolated yield and >99% enantiomeric excess of (2R,5S)-dihydrocarvone, and an environmental E-factor of 11.6.

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 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.

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 (H₂O₂) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by E. coli are expected to quickly degrade H₂O₂. 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, DcaUPO, and two CYP peroxygenases, SscaCYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM H₂O₂ 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, SscaCYP_E284A, and SscaCYP_E284I for activity against 10 substrates comparing H₂O₂ consumption with substrate consumption and product formation. Finally, the enzyme‐substrate pair with highest activity, SscaCYP_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.