Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase

Sumit Chakraborty, Natalia S. Nemeria, Anand Balakrishnan, Gabriel S. Brandt, Malea M. Kneen, Alejandra Yep, Michael J. McLeish, George L. Kenyon, Gregory A. Petsko, Dagmar Ringe, Frank Jordan

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with λ max at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with λ max at 437 nm. The time course of formation/ depletion of the PKB - ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB - ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with λ max at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 Å) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2α atom, rather than of the enamine, and supporting the assignment of the λ max at 473 nm to the PAA - ThDP adduct. The structure of the PAA - ThDP covalent complex is the first example of a product - ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

Original languageEnglish (US)
Pages (from-to)981-994
Number of pages14
JournalBiochemistry
Volume48
Issue number5
DOIs
StatePublished - Feb 10 2009

Fingerprint

benzoylformate decarboxylase
Thiamine Pyrophosphate
Decarboxylation
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Chakraborty, Sumit ; Nemeria, Natalia S. ; Balakrishnan, Anand ; Brandt, Gabriel S. ; Kneen, Malea M. ; Yep, Alejandra ; McLeish, Michael J. ; Kenyon, George L. ; Petsko, Gregory A. ; Ringe, Dagmar ; Jordan, Frank. / Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase. In: Biochemistry. 2009 ; Vol. 48, No. 5. pp. 981-994.
@article{2aa1059b86554233be527dc6ec911538,
title = "Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase",
abstract = "The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with λ max at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with λ max at 437 nm. The time course of formation/ depletion of the PKB - ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB - ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with λ max at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 {\AA}) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2α atom, rather than of the enamine, and supporting the assignment of the λ max at 473 nm to the PAA - ThDP adduct. The structure of the PAA - ThDP covalent complex is the first example of a product - ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.",
author = "Sumit Chakraborty and Nemeria, {Natalia S.} and Anand Balakrishnan and Brandt, {Gabriel S.} and Kneen, {Malea M.} and Alejandra Yep and McLeish, {Michael J.} and Kenyon, {George L.} and Petsko, {Gregory A.} and Dagmar Ringe and Frank Jordan",
year = "2009",
month = "2",
day = "10",
doi = "10.1021/bi801810h",
language = "English (US)",
volume = "48",
pages = "981--994",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",

}

Chakraborty, S, Nemeria, NS, Balakrishnan, A, Brandt, GS, Kneen, MM, Yep, A, McLeish, MJ, Kenyon, GL, Petsko, GA, Ringe, D & Jordan, F 2009, 'Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase', Biochemistry, vol. 48, no. 5, pp. 981-994. https://doi.org/10.1021/bi801810h

Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase. / Chakraborty, Sumit; Nemeria, Natalia S.; Balakrishnan, Anand; Brandt, Gabriel S.; Kneen, Malea M.; Yep, Alejandra; McLeish, Michael J.; Kenyon, George L.; Petsko, Gregory A.; Ringe, Dagmar; Jordan, Frank.

In: Biochemistry, Vol. 48, No. 5, 10.02.2009, p. 981-994.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase

AU - Chakraborty, Sumit

AU - Nemeria, Natalia S.

AU - Balakrishnan, Anand

AU - Brandt, Gabriel S.

AU - Kneen, Malea M.

AU - Yep, Alejandra

AU - McLeish, Michael J.

AU - Kenyon, George L.

AU - Petsko, Gregory A.

AU - Ringe, Dagmar

AU - Jordan, Frank

PY - 2009/2/10

Y1 - 2009/2/10

N2 - The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with λ max at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with λ max at 437 nm. The time course of formation/ depletion of the PKB - ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB - ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with λ max at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 Å) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2α atom, rather than of the enamine, and supporting the assignment of the λ max at 473 nm to the PAA - ThDP adduct. The structure of the PAA - ThDP covalent complex is the first example of a product - ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

AB - The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with λ max at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with λ max at 437 nm. The time course of formation/ depletion of the PKB - ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB - ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with λ max at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 Å) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2α atom, rather than of the enamine, and supporting the assignment of the λ max at 473 nm to the PAA - ThDP adduct. The structure of the PAA - ThDP covalent complex is the first example of a product - ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

UR - http://www.scopus.com/inward/record.url?scp=61449240107&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=61449240107&partnerID=8YFLogxK

U2 - 10.1021/bi801810h

DO - 10.1021/bi801810h

M3 - Article

C2 - 19140682

AN - SCOPUS:61449240107

VL - 48

SP - 981

EP - 994

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -