Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity

Buller, Andrew R.; Roye, P. van; Murciano-Calles, Javier; Arnold, Frances H.

doi:10.1021/acs.biochem.6b01127

Cited by 22 publications

(27 citation statements)

References 22 publications

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“…Natively, Trp synthase uses the cofactor pyridoxal phosphate (PLP) to condense l ‐serine and indole into Trp. Many substituted indoles and even other amino acids can participate in this versatile reaction, which affords access to complex starting materials for downstream modification . Recently, these efforts have been made even easier by evolving the β‐subunit of tryptophan synthase (TrpB; EC 4.2.1.20) for efficient stand‐alone function .…”

Section: Methodsmentioning

confidence: 99%

Facile in Vitro Biocatalytic Production of Diverse Tryptamines

2019

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Tryptamines are a medicinally important class of small molecules that serve as precursors to more complex, clinically used indole alkaloid natural products. Typically, tryptamine analogues are prepared from indoles through multistep synthetic routes. In the natural world, the desirable tryptamine synthon is produced in a single step by l‐tryptophan decarboxylases (TDCs). However, no TDCs are known to combine high activity and substrate promiscuity, which might enable a practical biocatalytic route to tryptamine analogues. We have now identified the TDC from Ruminococcus gnavus as the first highly active and promiscuous member of this enzyme family. RgnTDC performs up to 96 000 turnovers and readily accommodates tryptophan analogues with substituents at the 4, 5, 6, and 7 positions, as well as alternative heterocycles, thus enabling the facile biocatalytic synthesis of >20 tryptamine analogues. We demonstrate the utility of this enzyme in a two‐step biocatalytic sequence with an engineered tryptophan synthase to afford an efficient, cost‐effective route to tryptamines from commercially available indole starting materials.

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Section: Methodsmentioning

confidence: 99%

Facile in Vitro Biocatalytic Production of Diverse Tryptamines

2019

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“…The enzyme is composed of two catalytic units, TrpA ( subunit) and TrpB ( subunit), that associate into a functional heterotetrameric hydrolyase complex (TrpAB) (Dunn, 2012;Dunn et al, 2008;Raboni et al, 2003Raboni et al, , 2009. The enzyme catalyzes two reactions: the subunit converts 1-C-(indol-3-yl)glycerol 3-phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate, while the subunit condenses indole with l-serine to give l-tryptophan, with pyridoxal 5 0 -phosphate (PLP) functioning as a cofactor (Buller et al, 2016). In this process, the indole molecule is passed from subunit to the active site of subunit via an $25 Å tunnel to avoid indole leaking through the cell membranes (Hyde et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

“…The active site of subunit , which in the SpTrpA sequence comprises Glu52, Asp63, Tyr175, Gly213 and Gly234, catalyzes the retro-aldol cleavage of IGP using a push-pull general acid-base mechanism (Buller et al, 2016). The active-site pocket is highly conserved and is located in the center of the TIM barrel ( Supplementary Fig.…”

Section: Introductionmentioning

confidence: 99%

3D domain swapping in the TIM barrel of the α subunit ofStreptococcus pneumoniaetryptophan synthase

Michalska

Kowiel

Bigelow

et al. 2020

Acta Cryst Sect D Struct Biol

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Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0–S1–H1–S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2′. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α–β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.

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“…When indole glycerol-3-phosphate is used as the source of indole, the enzyme shows a >82,000-fold preference for Ser over Thr. 47…”

Section: Engineering Improved Ncaa Synthasesmentioning

confidence: 99%

Engineering enzymes for noncanonical amino acid synthesis

2018

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The standard proteinogenic amino acids grant access to a myriad of chemistries that harmonize to create life. Outside of these twenty canonical protein building blocks are countless noncanonical amino acids (ncAAs), either found in nature or created by man. Interest in ncAAs has grown as research has unveiled their importance as precursors to natural products and pharmaceuticals, biological probes, and more. Despite their broad applications, synthesis of ncAAs remains a challenge, as poor stereoselectivity and low functional-group compatibility stymie effective preparative routes. The use of enzymes has emerged as a versatile approach to prepare ncAAs, and nature’s enzymes can be engineered to synthesize ncAAs more efficiently and expand the amino acid alphabet. In this tutorial review, we briefly outline different enzyme engineering strategies and then discuss examples where engineering has generated new ‘ncAA synthases’ for efficient, environmentally benign production of a wide and growing collection of valuable ncAAs.

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Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity

Cited by 22 publications

References 22 publications

Facile in Vitro Biocatalytic Production of Diverse Tryptamines

Facile in Vitro Biocatalytic Production of Diverse Tryptamines

3D domain swapping in the TIM barrel of the α subunit ofStreptococcus pneumoniaetryptophan synthase

Engineering enzymes for noncanonical amino acid synthesis

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