Dataset Information

Stabilization of cyclohexanone monooxygenase by computational and experimental library design.

ABSTRACT: Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature-sensitive enzymes catalyzing the Baeyer-Villiger reaction, which display excellent stereo- and regioselectivity and offer a green alternative to the commonly used, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI-31, a substrate-promiscuous enzyme that efficiently catalyzes the production of the nylon-6 precursor ?-caprolactone. We used a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein-stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13°C and an approximately 33x increase in half-life at 30°C. In contrast to the wild-type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications.

SUBMITTER: Furst MJLJ

PROVIDER: S-EPMC6836875 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Stabilization of cyclohexanone monooxygenase by computational and experimental library design.

Fürst Maximilian J L J MJLJ Boonstra Marjon M Bandstra Selle S Fraaije Marco W MW

Biotechnology and bioengineering 20190624 9

Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature-sensitive enzymes catalyzing the Baeyer-Villiger reaction, which display excellent stereo- and regioselectivity and offer a green alternative to the commonly used, explos ...[more]

PMID: 31124128

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Project description:Members of the Sarbecovirus subgenus of Coronaviridae have twice caused deadly threats to humans. There is increasing concern about the rapid mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has evolved into multiple generations of epidemic variants in 3 years. Broad neutralizing antibodies are of great importance for pandemic preparedness against SARS-CoV-2 variants and divergent zoonotic sarbecoviruses. Here, we analyzed the structural conservation of the receptor-binding domain (RBD) from representative sarbecoviruses and chose S2H97, a previously reported RBD antibody with ideal breadth and resistance to escape, as a template for computational design to enhance the neutralization activity and spectrum. A total of 35 designs were purified for evaluation. The neutralizing activity of a large proportion of these designs against multiple variants was increased from several to hundreds of times. Molecular dynamics simulation suggested that extra interface contacts and enhanced intermolecular interactions between the RBD and the designed antibodies are established. After light and heavy chain reconstitution, AI-1028, with five complementarity determining regions optimized, showed the best neutralizing activity across all tested sarbecoviruses, including SARS-CoV, multiple SARS-CoV-2 variants, and bat-derived viruses. AI-1028 recognized the same cryptic RBD epitope as the parental prototype antibody. In addition to computational design, chemically synthesized nanobody libraries are also a precious resource for rapid antibody development. By applying distinct RBDs as baits for reciprocal screening, we identified two novel nanobodies with broad activities. These findings provide potential pan-sarbecovirus neutralizing drugs and highlight new pathways to rapidly optimize therapeutic candidates when novel SARS-CoV-2 escape variants or new zoonotic coronaviruses emerge. IMPORTANCE The subgenus Sarbecovirus includes human SARS-CoV, SARS-CoV-2, and hundreds of genetically related bat viruses. The continuous evolution of SARS-CoV-2 has led to the striking evasion of neutralizing antibody (NAb) drugs and convalescent plasma. Antibodies with broad activity across sarbecoviruses would be helpful to combat current SARS-CoV-2 mutations and longer term animal virus spillovers. The study of pan-sarbecovirus NAbs described here is significant for the following reasons. First, we established a structure-based computational pipeline to design and optimize NAbs to obtain more potent and broader neutralizing activity across multiple sarbecoviruses. Second, we screened and identified nanobodies from a highly diversified synthetic library with a broad neutralizing spectrum using an elaborate screening strategy. These methodologies provide guidance for the rapid development of antibody therapeutics against emerging pathogens with highly variable characteristics.

Dataset Information

Stabilization of cyclohexanone monooxygenase by computational and experimental library design.

Publications

Stabilization of cyclohexanone monooxygenase by computational and experimental library design.

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