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Substrate Channeling by a Rationally Designed Fusion Protein in a Biocatalytic Cascade.

ABSTRACT: Substrate channeling, where an intermediate in a multistep reaction is directed toward a reaction center rather than freely diffusing, offers several advantages when employed in catalytic cascades. Here we present a fusion enzyme comprised of an alcohol and aldehyde dehydrogenase, that is computationally designed to facilitate electrostatic substrate channeling using a cationic linker bridging the two structures. Rosetta protein folding software was utilized to determine an optimal linker placement, added to the truncated termini of the proteins, which is as close as possible to the active sites of the enzymes without disrupting critical catalytic residues. With improvements in stability, product selectivity (90%), and catalyst turnover frequency, representing 500-fold increased activity compared to the unbound enzymes and nearly 140-fold for a neutral-linked fusion enzyme, this design strategy holds promise for making other multistep catalytic processes more sustainable and efficient.

SUBMITTER: Kummer MJ

PROVIDER: S-EPMC8397353 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Substrate Channeling by a Rationally Designed Fusion Protein in a Biocatalytic Cascade.

Kummer Matthew J MJ Lee Yoo Seok YS Yuan Mengwei M Alkotaini Bassam B Zhao John J Blumenthal Emmy E Minteer Shelley D SD

JACS Au 20210701 8

Substrate channeling, where an intermediate in a multistep reaction is directed toward a reaction center rather than freely diffusing, offers several advantages when employed in catalytic cascades. Here we present a fusion enzyme comprised of an alcohol and aldehyde dehydrogenase, that is computationally designed to facilitate electrostatic substrate channeling using a cationic linker bridging the two structures. Rosetta protein folding software was utilized to determine an optimal linker placem ...[more]

PMID: 34467357

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Project description:BackgroundChitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated.ResultsHerein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure.ConclusionsUsing oxidase-amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.

Dataset Information

Substrate Channeling by a Rationally Designed Fusion Protein in a Biocatalytic Cascade.

Publications

Substrate Channeling by a Rationally Designed Fusion Protein in a Biocatalytic Cascade.

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