Asymmetric Alkylation of Ketones Catalyzed by Engineered TrpB

Watkins‐Dulaney, Ella J.; Dunham, N.P.; Straathof, Sabine; Turi, Soma; Arnold, Frances H.; Buller, Andrew R.

doi:10.1002/ange.202106938

Cited by 3 publications

(4 citation statements)

References 29 publications

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“…TrpB natively catalyzes β‐substitution of the hydroxyl side chain of serine for indole through an electrophilic amino acrylate intermediate, E(A−A). Inspired by this highly selective C−C bond forming reaction, researchers have subjected TrpB to extensive engineering for β‐substitution with a variety of C ‐nucleophiles including indoles, nitroalkanes, and enols [14,15,18,23] . These TrpB enzymes often react less efficiently with S ‐ and N ‐nucleophiles, and none are known to catalyze β‐replacement with O ‐nucleophiles [19,24,25] .…”

Section: Introductionmentioning

confidence: 99%

“…Inspired by this highly selective CÀ C bond forming reaction, researchers have subjected TrpB to extensive engineering for β-substitution with a variety of C-nucleophiles including indoles, nitroalkanes, and enols. [14,15,18,23] These TrpB enzymes often react less efficiently with S-and N-nucleophiles, and none are known to catalyze β-replacement with Onucleophiles. [19,24,25] Given that the products of such reactions are desirable building blocks, we considered the synthetic potential of other enzymes from the fold type II family.…”

Section: Introductionmentioning

confidence: 99%

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Investigation of β‐Substitution Activity of O‐Acetylserine Sulfhydrolase from Citrullus vulgaris

et al. 2022

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Pyridoxal-5'-phosphate (PLP)-dependent enzymes have garnered interest for their ability to synthesize non-standard amino acids (nsAAs). One such class of enzymes, O-acetylserine sulfhydrylases (OASSs), catalyzes the final step in the biosynthesis of l-cysteine. Here, we examine the β-substitution capability of the OASS from Citrullus vulgaris (CvOASS), a putative l-mimosine synthase. While the previously reported mimosine synthase activity was not reproducible in our hands, we successfully identified non-native reactivity with a variety of O-nucleophiles. Optimization of reaction conditions for carbox-ylate and phenolate substrates led to distinct conditions that were leveraged for the preparative-scale synthesis of nsAAs. We further show this enzyme is capable of CÀ C bond formation through a β-alkylation reaction with an activated nitroalkane. To facilitate understanding of this enzyme, we determined the crystal structure of the enzyme bound to PLP as the internal aldimine at 1.55 Å, revealing key features of the active site and providing information that may guide subsequent development of CvOASS as a practical biocatalyst.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Investigation of β‐Substitution Activity of O‐Acetylserine Sulfhydrolase from Citrullus vulgaris

et al. 2022

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“…These data provoked us to consider an alternative, simpler hypothesis: proton transfers to conjugated C-bases are intrinsically slower due to high re-organization energies. 35,36 In this scenario, the 'kinetic enolate' reactivity of ObiH is not due to some special evolutionary selective pressure, but rather the absence of one. Although PLP alone enables slow proton transfer at Cα, these events are nevertheless sluggish, and enzymes must evolve to accelerate them.…”

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Biocatalytic asymmetric aldol addition into unactivated ketones

Bruffy,

Meza,

Soler

et al. 2023

Preprint

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Aldolases are prodigious C-C bond forming enzymes, but their reactivity has only been extended past activated carbonyl electrophiles in special cases. We have used a pair of pyridoxal-phosphate-dependent aldolases to probe the mechanistic origins of this limitation. Our results reveal how aldolases are limited by thermodynamically favorable proton transfer with solvent, which undermines aldol addition into ketones. However, we show how a transaldolase can circumvent this limitation by protecting the enzyme-bound enolate from solvent protons and thereby enabling efficient addition into unactivated ketones. The resulting products are non-canonical amino acids with side chains that contain chiral tertiary alcohols. This study reveals the principles for extending aldolase catalysis beyond its previous limits and enables convergent, enantioselective C-C bond formation from simple starting materials.

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“…For example, pyridoxal phosphate (PLP)-dependent enzymes are preeminent for the manipulation of amino acids, and many have found industrial use. , The broad utility of non-canonical amino acids (ncAAs) in medicinal chemistry , and chemical and synthetic biology − has spurred a range of biocatalytic strategies to synthesize them. − Of particular utility are enzymes that can activate an amino acid donor molecule to react with a range of substrate partners in a convergent and stereoselective manner. Some native PLP-dependent enzymes satisfy these criteria and operate on preparative scales to yield a variety of substituted ncAAs. − When native enzymatic activity is too low, directed evolution can be used to improve the activity of convergent, complexity-building ncAA synthases. − Recently, PLP-dependent enzymes from outside central metabolism were found to catalyze γ-substitution en route to a variety of structurally diverse ncAAs. − Enzymes that catalyze γ-substitution (called γ-synthases) operate through a common vinylglycine ketimine (VGK) intermediate (Figure A). Currently, it is not known which γ-synthases have desirable substrate promiscuity or which ones suffer from parasitic reaction pathways like hydrolysis.…”

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Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family

Zmich,

Perkins,

Bingman

et al. 2023

ACS Catal.

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Pyridoxal phosphate (PLP)-dependent enzymes afford access to a variety of non-canonical amino acids (ncAAs), which are premier building blocks for the construction of complex bioactive molecules. The vinylglycine ketimine (VGK) subfamily of PLPdependent enzymes plays a critical role in sulfur metabolism and is home to a growing set of secondary metabolic enzymes that synthesize γ-substituted ncAAs. Identification of VGK enzymes for biocatalysis faces a distinct challenge because the subfamily contains both desirable synthases and lyases that break down ncAAs. Some enzymes have both activities, which may contribute to pervasive mis-annotation. To navigate this complex functional landscape, we used a substrate multiplexed screening approach to rapidly measure the substrate promiscuity of 40 homologs in the VGK subfamily. We found that enzymes involved in transsulfuration are less likely to have promiscuous activities and often possess undesirable lyase activity. Enzymes from direct sulfuration and secondary metabolism generally had a high degree of substrate promiscuity. From this cohort, we identified an exemplary γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). This enzyme is thermostable and has high expression (∼400 mg protein per L culture), enabling preparative-scale synthesis of thioether containing ncAAs. When assayed with L-allylglycine, CahyGS catalyzes a stereoselective γ-addition reaction to afford access to a unique set of γ-methyl-branched ncAAs. We determined high-resolution crystal structures of this enzyme that define an open-close transition associated with ligand binding and set the stage for future engineering within this enzyme subfamily.

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Asymmetric Alkylation of Ketones Catalyzed by Engineered TrpB

Abstract: Supporting information for this article is given via a link at the end of the document.

Cited by 3 publications

References 29 publications

Investigation of β‐Substitution Activity of O‐Acetylserine Sulfhydrolase from Citrullus vulgaris

Investigation of β‐Substitution Activity of O‐Acetylserine Sulfhydrolase from Citrullus vulgaris

Biocatalytic asymmetric aldol addition into unactivated ketones

Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family

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