Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

McDonald, Allwin D.; Higgins, Peyton; Buller, Andrew R.

doi:10.1038/s41467-022-32789-w

Cited by 19 publications

(13 citation statements)

References 63 publications

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“…Despite this, only recently have research efforts recognized generality as a target property to be optimized for (e.g., yield, selectivity, etc.) with few examples in transition metal-, , bio-, and photocatalysis . Regarding asymmetric catalysis, recent works have focused on using high-throughput techniques allowing for direct comparative studies of catalyst performance. , While such protocols assess an important aspect of generality, they do not capture the impact of the catalyst structure in a high-dimensional search space.…”

Section: Introductionmentioning

confidence: 99%

A Data-Driven Workflow for Assigning and Predicting Generality in Asymmetric Catalysis

et al. 2023

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The development of chiral catalysts that can provide high enantioselectivities across a wide assortment of substrates or reaction range is a priority for many catalyst design efforts. While several approaches are available to aid in the identification of general catalyst systems, there is currently no simple procedure for directly measuring how general a given catalyst could be. Herein, we present a catalyst-agnostic workflow centered on unsupervised machine learning that enables the rapid assessment and quantification of catalyst generality. The workflow uses curated literature data sets and reaction descriptors to visualize and cluster chemical space coverage. This reaction network can then be applied to derive a catalyst generality metric through designer equations and interfaced with other regression techniques for general catalyst prediction. As validating case studies, we have successfully applied this method to identify-through-quantification the most general catalyst chemotype for an organocatalytic asymmetric Mannich reaction and predicted the most general chiral phosphoric acid catalyst for the addition of nucleophiles to imines. The mechanistic basis for catalyst generality can then be gleaned from the calculated values by deconstructing the contributions of chemical space and enantiomeric excess to the overall result. Finally, our generality techniques permitted the development of mechanistically informative catalyst screening sets that allow experimentalists to rationally select catalysts that have the highest probability of achieving a good result in the first round of reaction development. Overall, our findings represent a framework for interrogating catalyst generality, and this strategy should be relevant to other catalytic systems widely applied in asymmetric synthesis.

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Section: Introductionmentioning

confidence: 99%

A Data-Driven Workflow for Assigning and Predicting Generality in Asymmetric Catalysis

et al. 2023

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“…This substrate multiplexed screening (SUMS) approach offers several distinct advantages compared to single substrate measurements. 34,35 Comparison of relative rates of product formation from single-substrate reactions provides only indirect information about specificity. By assaying multiple substrates in competition, SUMS gives direct information on the ability of an enzyme to discriminate between two or more substrates.…”

Section: Resultsmentioning

confidence: 99%

Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Zmich

Perkins

Bingman

et al. 2023

Preprint

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Sequence-based functional annotation of enzymes is an essential step in the discovery and development of new biocatalysts. The vinylglycine ketimine (VGK) subfamily of pyridoxal-phosphate dependent enzymes plays critical roles in sulfur metabolism and is home to a growing range of secondary metabolic enzymes that synthesize noncanonical amino acids. However, discovery of useful new enzymes has been slowed because functional assignments within the VGK sub-family are convoluted by pervasive mis-annotation. Here, we used a whole-cell substrate multiplexed screening approach to rapidly measure catalytic activities of 40 homologs in the VGK subfamily. This strategy gives direct information on enzyme specificity without having to purify each enzyme or measure individual kinetic constants. We identified a thermostable cystathionine γ-lyase from Thermobifida fusca and performed mechanistic and structural studies. For biocatalytic applications, we identified a well-behaved, thermostable, and promiscuous amino acid γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). We showed CahyGS can catalyze a stereoselective γ-addition into L-allylglycine, providing preparative-scale access to a unique set of γ-branched amino acids. High resolution crystal structures of CahyGS show an open-closed transition associated with ligand binding and provide a basis for subsequent engineering. Together, these data show how multiplexed screening approaches aid in the rapid deconvolution of enzyme function and identify enzymes with useful properties for enzymology and biocatalysis.

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“…Previously, Substrate Multiplexed Screening (SUMS) has been demonstrated to assess enzyme promiscuity, wherein multiple ligands competed for an enzyme. [19] With a similar approach, we wanted to assess whether the preferences of MTases for DM‐AdoMets would allow selective modification of a target molecule in a mixture of substrates and MTases. In our case, two enzymes were competed for a common substrate.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic Generation of Double‐Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases

et al. 2022

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Methyltransferases (MTases) have become an important tool for site‐specific alkylation and biomolecular labelling. In biocatalytic cascades with methionine adenosyltransferases (MATs), transfer of functional moieties has been realized starting from methionine analogues and ATP. However, the widespread use of S‐adenosyl‐l‐methionine (AdoMet) and the abundance of MTases accepting sulfonium centre modifications limit selective modification in mixtures. AdoMet analogues with additional modifications at the nucleoside moiety bear potential for acceptance by specific MTases. Here, we explored the generation of double‐modified AdoMets by an engineered Methanocaldococcus jannaschii MAT (PC‐MjMAT), using 19 ATP analogues in combination with two methionine analogues. This substrate screening was extended to cascade reactions and to MTase competition assays. Our results show that MTase targeting selectivity can be improved by using bulky substituents at the N6 of adenine. The facile access to >10 new AdoMet analogues provides the groundwork for developing MAT‐MTase cascades for orthogonal biomolecular labelling.

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Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Cited by 19 publications

References 63 publications

A Data-Driven Workflow for Assigning and Predicting Generality in Asymmetric Catalysis

A Data-Driven Workflow for Assigning and Predicting Generality in Asymmetric Catalysis

Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Enzymatic Generation of Double‐Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases

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