Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from
            <i>Saccharomyces cerevisiae</i>

Fiedler, Erik; Thorell, Stina; Sandalova, T.; Golbik, Ralph; König, Stephan; Schneider, Günter

doi:10.1073/pnas.022510999

Cited by 125 publications

(157 citation statements)

References 32 publications

Supporting

Mentioning

141

Contrasting

Unclassified

Order By: Relevance

“…His26 and His66 are both directly involved in catalysis and the substrate specificity of TK , and are found to be highly conserved in all TK-like enzymes. His100 is conserved in all TK-like enzymes except DXPS, agreeing with an earlier report (Fiedler et al 2002).…”

Section: Alignment and Conservation Within Pp-and Pyr-domainssupporting

confidence: 92%

See 1 more Smart Citation

Evolutionary Analysis of the TPP-Dependent Enzyme Family

2007

View full text Add to dashboard Cite

The evolutionary relationships of the thiamine pyrophosphate (TPP) dependent family of enzymes was investigated by generation of a neighbor joining (NJ) phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes.This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood (ML) trees generated for each individual enzyme. The study discerned the order in which enzymes diverged in the progenote population and several instances where enzyme types diverged later, and broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The relationship between the PP-and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC) domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity towards β-hydroxypyruvate (HPA) and glycolaldehyde (GA). Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP-and Pyr-domains whose evolutionary histories extend to all TPP-dependent enzymes.

show abstract

Section: Alignment and Conservation Within Pp-and Pyr-domainssupporting

confidence: 92%

“…This residue is highly conserved across TK, DXPS and PKL, but less conserved in the other enzyme types. Glu477 (ScePDC) is similarly involved in metal-ion binding and is essential for catalysis in PDC (Fiedler et al 2002). …”

Section: Alignment and Conservation Within Pp-and Pyr-domainsmentioning

confidence: 99%

Evolutionary Analysis of the TPP-Dependent Enzyme Family

2007

View full text Add to dashboard Cite

show abstract

“…In tyrosine phenol lyase, an aspartate group is positioned close to the pyridine nitrogen, poised to stabilize a quinonoid species, and the ϵ-amino group of the active site lysine is 3.7 Å away from Cα. The only other carbanion species that has been characterized crystallographically is the α-carbanion/enamine of thiamine diphosphate in transketolase (35). We expect the mechanism of carbanion stabilization by a multipurpose Schiff base-forming lysine to be common to other PLP enzymes that do not depend on the formation of a quinonoid intermediate.…”

Section: Discussionmentioning

confidence: 99%

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.

101

247

View full text Add to dashboard Cite

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.

show abstract

“…It has been widely assumed that this delocalization is a necessary component of the reaction pathway. In fact, crystallographic analysis suggested the presence of an accumulation of resonance-stabilized, enaminetype intermediates in some, but not all, ThDP enzymes; however, the structural resolution (1.8-2.5 Å) precluded an unambiguous assignment in all these instances (5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin

Meyer

Neumann

Koers

et al. 2012

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

View full text Add to dashboard Cite

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.crystallography | tautomerization | enzyme mechanism

show abstract

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Cited by 125 publications

References 32 publications

Evolutionary Analysis of the TPP-Dependent Enzyme Family

Evolutionary Analysis of the TPP-Dependent Enzyme Family

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

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin

Contact Info

Product

Resources

About