Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

Barends, Thomas R. M.; Domratcheva, Tatiana; Kulik, V.; Blumenstein, Lars; Niks, Dimitri; Dunn, Michael F.; Schlichting, Ilme

doi:10.1002/cbic.200700703

Cited by 50 publications

(99 citation statements)

References 16 publications

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“…A mixture of a quinonoid species and other intermediates has been reported in the 2.9-Å crystal structure of PLPdependent serine hydroxymethyltransferase, in which retro-aldol cleavage of the Cα-Cβ bond rather than proton abstraction from Cα generates a quinonoid intermediate (32). Quinonoid intermediates have also been reported in the structures of tyrosine phenol lyase in the presence of substrate analogs (33) and in tryptophan synthase in the presence of a substrate mimic (34). Although the quinonoid species obtained with a substrate mimic in the tryptophan synthase structure is not directly comparable to the serine-derived intermediate in dCBS, it exhibits a similar Cα geometry.…”

Section: Discussionmentioning

confidence: 86%

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

Koutmos

Kabil

Smith

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

104

247

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The catalytic potential for H 2 S biogenesis and homocysteine clearance converge at the active site of cystathionine β-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes β-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H 2 S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 Å) and an aminoacrylate intermediate (1.55 Å) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory “energy-sensing” CBS domains, named after this protein, suggests a mechanism for allosteric activation by S -adenosylmethionine.

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Section: Discussionmentioning

confidence: 86%

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

Koutmos

Kabil

Smith

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

104

247

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“…bLys87 has been proposed as the active site base involved in the abstraction of the a-proton of the external aldimine and in the protonation of the tryptophan quinonoid intermediate prior to release of L-tryptophan from its external aldimine. Solution studies [58,60], including the determination of steady state and pre-steady state isotope effects as a function of pH, in the absence and presence of monovalent cations [15], as well as recent structural work [16,31], have provided strong evidence supporting bGlu109 as the proton acceptor from the a-amine of L-serine and the proton donor to the leaving hydroxide in stage I (Fig. 2).…”

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 98%

“…In the b-subunit, the stabilization of alternative catalytic intermediates and conformations is mediated by the interaction between bArg141 and bAsp305 (Fig. 2) [16,31,61,62,95]. In the internal aldimine (Fig.…”

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 99%

“…More recently, a new family of unreactive a-site ligands, which contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups, were proven to bind to the a-site with high specificities and affinities, mimicking the complex between IGP and G3P and acting, as did the previously discovered IPP or IAA, as allosteric ligands. They were reported to slow the entry of indole analogues into the b-site by blocking the tunnel opening at the a-site and stabilizing the closed conformation of the b-subunit [16,31].…”

Section: Catalytic Mechanism and Regulatory Effectsmentioning

confidence: 99%

“…This information is coupled to a series of X-ray crystallographic studies, pioneered by Davies, Miles and co-workers [12] and later pursued by Schlichting, Mozzarelli, Dunn and co-workers [13], that led to a deep understanding of structure-function relationships [13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Tryptophan synthase: a mine for enzymologists

Raboni

Bettati

Mozzarelli

2009

Cell. Mol. Life Sci.

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Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the last two steps of tryptophan biosynthesis in bacteria, plants and fungi. Structural, dynamic and functional studies, carried out over more than 40 years, have unveiled that: (1) alpha- and beta-active sites are separated by about 20 A and communicate via the selective stabilization of distinct conformational states, triggered by the chemical nature of individual catalytic intermediates and by allosteric ligands; (2) indole, formed at alpha-active site, is intramolecularly channeled to the beta-active site; and (3) naturally occurring as well as genetically generated mutants have allowed to pinpoint functional and regulatory roles for several individual amino acids. These key features have made tryptophan synthase a text-book case for the understanding of the interplay between chemistry and conformational energy landscapes.

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Engineered Biocatalytic Synthesis of β‐N‐Substituted‐α‐Amino Acids

Villalona,

Higgins,

Buller

2023

Angewandte Chemie

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Non‐canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for synthesis of β‐substituted amino acids. However, there are few examples of biocatalytic N‐substitution reactions to make α,β‐diamino acids. Here, we use directed evolution to engineer the β‐subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re‐entry into the catalytic cycle and subsequent decomposition. Additional equivalents of L‐serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β‐substitution with a dozen aryl amine nucleophiles, including demonstration on a g‐scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.

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Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

Cited by 50 publications

References 16 publications

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

Tryptophan synthase: a mine for enzymologists

Engineered Biocatalytic Synthesis of β‐N‐Substituted‐α‐Amino Acids

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