Engineered Biosynthesis of β‐Alkyl Tryptophan Analogues

Boville, Christina E.; Scheele, Remkes A.; Koch, Philipp; Brinkmann‐Chen, Sabine; Buller, Andrew R.; Arnold, Frances H.

doi:10.1002/anie.201807998

Cited by 59 publications

(63 citation statements)

References 36 publications

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“…2 We evolved this enzyme to accept a variety of indole analogues and nitroalkanes with activated sp³-carbon atoms as nucleophiles ( Figure 1b). [3][4][5][6][7][8] However, these studies revealed limitations including chemo-and stereoselective control we would need to address in order to realize TrpB's broad potential as a biocatalyst for C-C bond formation. Previous biocatalytic reactions catalyzed by variants of TrpB [5][6]8 and proposed stereo-and chemo-selective reaction using engineered TrpB.…”

Section: Introductionmentioning

confidence: 99%

“…2,10 TrpB is highly stereoselective and can retain the absolute configuration of the Cα of L-serine after product formation (Figure 1b). [3][4][5][6][7] However, the stereoselectivity at potential new stereogenic centers is unknown. Substrates with nucleophilic sp 3 -hybridized carbons have the ability to form a new quaternary carbon at the γ-position.…”

Section: Introductionmentioning

confidence: 99%

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Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

et al. 2019

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We previously engineered the tryptophan synthase b-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-tryptophan, to synthesize a range of modified tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form CC bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking the oxindole N1 atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant Pfquat efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L E. coli culture, TrpB Pfquat provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

et al. 2019

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“…[66] Christina Boville and team engineered stand-alone PfTrpB 2B9 to accept β-branched Ser analogs with longer alkyl chains such as β-ethyl-and β-propylSer. [67] Bulkier alkyl chains at the β-position were thought to hinder nucleophilic attack, allowing the competing β-elimination to unproductively consume the electrophile. Initial activity with PfTrpB 2B9 on β-ethylSer and indole was too low for high-throughput screening, so the authors mutated an active-site residue presumed to clash sterically with the alkyl β-substitution.…”

Section: β-Branched Trpsmentioning

confidence: 99%

“…X-ray crystallography, measurement of the deamination rates and UVspectrophotometric evidence supported the hypothesis that evolution stabilized the closed conformation of the enzyme and generated a more persistent amino-acrylate that was less prone to unproductive β-elimination . [67]…”

Section: β-Branched Trpsmentioning

confidence: 99%

Tryptophan Synthase: Biocatalyst Extraordinaire

2020

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Tryptophan synthase (TrpS) has emerged as a paragon of noncanonical amino acid (ncAA) synthesis and is an ideal biocatalyst for synthetic and biological applications. TrpS catalyzes an irreversible, CC bond forming reaction between indole and serine (Ser) to make L-tryptophan (Trp); native TrpS complexes possess fairly broad specificity for indole analogs, but are difficult to engineer to extend substrate scope or to confer other useful properties due to allosteric constraints and their heterodimeric structure. Directed evolution freed the catalytically relevant TrpS β-subunit (TrpB) from allosteric regulation by its TrpA partner and has enabled dramatic expansion of the enzyme's substrate scope. This review examines the long and storied career of TrpS from the perspective of its application in ncAA synthesis and biocatalytic cascades.

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“…4.2.1.20) is a pyridoxal 5‐phosphate (PLP)‐dependent enzyme that catalyzes the synthesis of l ‐Trp from indole and l ‐Ser . Frances H. Arnold et al has reported that TrpS could react with myriad indole analogs to provide a direct biocatalytic route to l ‐Trp derivatives, a kind of l ‐ncAAs …”

Section: Introductionmentioning

confidence: 99%

Dynamic Kinetic Resolution for Asymmetric Synthesis of L‐Noncanonical Amino Acids from D‐Ser Using Tryptophan Synthase and Alanine Racemase

Gao

et al. 2019

Eur J Org Chem

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L‐Ser is often used to synthesize some significant l‐noncanonical α‐amino acids(l‐ncAAs), which are the prevalent intermediates and precursors for functional synthetic compounds. In this study, threonine aldolase from Escherichia coli k‐12 MG1655 has been used to synthesize l‐Ser. In contrast to the maximum catalytic capacity (20 g/L) for l‐threonine aldolase(LTA), d‐Ser was synthesized with high yield (240 g/L) from cheap Gly and paraformaldehyde using d‐threonine aldolase (DTA) from Arthrobacter sp ATCC. In order to fully utilize d‐Ser and expand the resource of l‐Ser, a dynamic kinetic resolution system was constructed to convert d/dl‐Ser to l‐Ser through combining alanine racemase (Alr) from Bacillus subtilis with l‐tryptophan synthase (TrpS) from Escherichia coli k‐12 MG1655, and l‐ncAAs including l‐Trp and l‐Cys derivatives were synthesized with excellent enantioselectivity and in high yields. The results indicated l‐ncAAs could be efficiently synthesized from d‐Ser using this original and green dynamic kinetic resolution system, and the reliable l‐Ser resource has been established from simple and achiral substrates.

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Engineered Biosynthesis of β‐Alkyl Tryptophan Analogues

Cited by 59 publications

References 36 publications

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Tryptophan Synthase: Biocatalyst Extraordinaire

Dynamic Kinetic Resolution for Asymmetric Synthesis of L‐Noncanonical Amino Acids from D‐Ser Using Tryptophan Synthase and Alanine Racemase

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