Catalytic Performance of a Class III Old Yellow Enzyme and Its Cysteine Variants

Abstract

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81–84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

Asymmetric Reduction of (R)‐Carvone through a Thermostable and Organic‐Solvent‐Tolerant Ene‐Reductase

“…From these data sets, the following order of catalytic efficiency can be deduced (under substrate saturation conditions and with BNAH as electron donor): XenA (≈1860 s −1 m m −1 )> F OYE‐1 (≈20 %)>OYE Ro 2a (≈17 %)>PETNR≈TOYE (4 %). With respect to turnover frequency, F OYE‐1 is the most powerful ER of the set ( k app four to 40 times higher than those of the other ERs listed) …”

“…In that work the enzyme performance with the natural nicotinamide electron donors in comparison with synthetic mimics was determined under substrate saturation (cyclohex‐2‐en‐1‐one) conditions at 30 °C. Similar experiments were performed with OYE Ro 2a and use of N ‐methylmaleimide ( 2 ) as the substrate at 25 °C . From these data sets, the following order of catalytic efficiency can be deduced (under substrate saturation conditions and with BNAH as electron donor): XenA (≈1860 s −1 m m −1 )> F OYE‐1 (≈20 %)>OYE Ro 2a (≈17 %)>PETNR≈TOYE (4 %).…”

“…Chemicals : NADPH and NADH were purchased from Prozomix. BNAH was freshly synthesized as described previously and assessed for stability by UV/Vis spectroscopy prior to each application . All other chemicals were purchased from Sigma–Aldrich or TCI Europe (Belgium or Germany) at highest purity available.…”

Chlamydomonas reinhardtii mutants deficient for Old Yellow Enzyme 3 exhibit increased photooxidative stress

“…Maleimides and maleimide‐like compounds have been detected as degradation products of Chl and heme, both in living organisms and in sediments (Gaubert et al, 2019 ; Naeher et al, 2013 ; Ritter et al, 2020 ; Suzuki & Shioi, 1999 ). Maleimides are toxic to cells because they react readily with the thiol groups of cysteines (Gunnoo & Madder, 2016 ; Scholtissek et al, 2018 ). Although, to the best of our knowledge, maleimides have not been detected in C. reinhardtii , it is well possible that the soil‐dwelling microalga is exposed to these degradation products from internal or external sources, making a dedicated defense mechanism such as their conversion by an ene‐reductase necessary.…”

Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

“…These cofactors act as hydride acceptor and donor intermediates between the substrate and product.Enzyme recognition of NAD(P), usually by a cofactor (binding) motif such as the Rossmann fold, is important for cellular processes, however when using in vitro systems, the adenine dinucleotide moiety of the cofactor becomes obsolete and disadvantageous, being prone to hydrolysis [4]. Synthetic nicotinamide coenzyme biomimetics (NCBs) have been shown to replace NAD(P)H in flavin-dependent enzymes such as nitroreductase and NAD(P)H quinone oxidoreductase [5-7], a cytochrome P450 BM3 variant [8], ene-reductases [9][10][11][12][13][14][15][16], and styrene monooxygenase [17]. In all cases described, a flavin was involved as an electron mediator [18][19][20][21][22].Jones pioneered the use of synthetic cofactor analogues with horse liver ADH (HLADH) [23][24][25][26], followed by Fish [27] and others [28,29].…”