Exploiting the Catalytic Diversity of Short‐Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope

Roth, Sebastian; Kilgore, Matthew B.; Kutchan, Toni M.; Müller, Michael

doi:10.1002/cbic.201800291

Cited by 22 publications

(31 citation statements)

References 31 publications

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“…Inspired by the unique active site compositions of NR and Zt _SDR, four mutations transformed an unrelated SDR with distinct keto‐reducing activity into a promiscuous enzyme with additional imine‐reducing activity. In addition to our previous results and literature examples, this highlights that a strict border splitting “ketoreductases” and “imine reductases” can be misleading.…”

Section: Methodssupporting

confidence: 70%

“…aff. pseudonarcissus and Zt _SDR from Zephyranthes treatiae . NR was originally identified as an enone reductase (C=C reduction) involved in alkaloid biosynthesis, highlighting the versatility of SDRs.…”

Section: Methodsmentioning

confidence: 99%

“… Stages of SDR‐catalyzed imine reduction. The serendipitous identification of iminium‐reducing activity of glucose dehydrogenase (GDH); exploitation of the catalytic promiscuity of noroxomaritidine reductase (NR); generation of imine‐reducing activity in an unrelated SDR by mutagenesis (this study).…”

Section: Methodsmentioning

confidence: 99%

“…A “common” BLAST resulted in hits with 60–70 % overall sequence identity. From these hits, an SDR from Asparagus officinalis ( Ao _SDR, 66 % sequence identity) was investigated . Despite the elevated sequence identity, Ao _SDR preferred keto substrates and featured only a negligible imine‐reducing activity.…”

Section: Methodsmentioning

confidence: 99%

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Crossing the Border: From Keto‐ to Imine Reduction in Short‐Chain Dehydrogenases/Reductases

et al. 2020

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The family of NAD(P)H‐dependent short‐chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto‐reducing activity, yet negligible imine‐reducing capability, and mining the Short‐Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active‐site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine‐reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto‐reducing activity was engineered into a promiscuous enzyme with imine‐reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine‐reducing enzymes.

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Section: Methodssupporting

confidence: 70%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Crossing the Border: From Keto‐ to Imine Reduction in Short‐Chain Dehydrogenases/Reductases

et al. 2020

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“…Both ZSD1 and ABA2 belong to plant SDR superfamily enzymes, which are the most important terpene modifying enzymes [35,36]. These SDRs are generally NAD(P)-dependent enzymes involved in secondary metabolism and display wide and promiscuous substrate specificity in catalyzing the oxidation of alcohol, sterol, saccharides, and aromatic compounds [40]. While ZSD1 has been biochemically characterized to catalyze the oxidation of sesquiterpene alcohol 8-hydroxy-α-humulene and monoterpene alcohol borneol in a NAD-dependent manner [36], ABA2 has been reported to be involved in the sesquiterpenoid ABA synthesis and to catalyze the oxidation of xanthoxin to abscisic aldehyde [35].…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone

et al. 2020

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Background: (+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield. Results: As the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively. Conclusion: We successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

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Biokatalytische Reduktionen aus der Sicht eines Chemikers

Hollmann

Opperman²,

Paul

2020

Angewandte Chemie

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Reduktionsreaktionen spielen eine zentrale Rolle in der organischen Chemie zur Synthese chiraler Produkte. Insbesondere Enzyme katalysieren solche Reaktionen in hoher Stereo‐, Regio‐ und Chemoselektivität. Somit eröffnen sie kürzere Alternativrouten zur Synthese von Spezial‐ und Bulkchemikalien. Dieser Aufsatz beschreibt den aktuellen Stand, die Herausforderungen und Möglichkeiten enzymkatalysierter Reduktionen von (konjugierten) Carbonylverbindungen, Aromaten sowie der reduktiven Aminierung.

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Exploiting the Catalytic Diversity of Short‐Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope

Cited by 22 publications

References 31 publications

Crossing the Border: From Keto‐ to Imine Reduction in Short‐Chain Dehydrogenases/Reductases

Crossing the Border: From Keto‐ to Imine Reduction in Short‐Chain Dehydrogenases/Reductases

Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone

Biokatalytische Reduktionen aus der Sicht eines Chemikers

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