One substrate, five products: Reactions catalyzed by the dihydroneopterin aldolase from Mycobacterium tuberculosis

Clarissa M. Czekster, John S. Blanchard

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Tetrahydrofolate cofactors are required for one carbon transfer reaction involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form a protein-mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild-type DHNAs. The enzyme-substrate complex partitions 51% in the first turnover to form the aldolase products, 24% to the epimerase product and 25% to the oxygenase products. The aldolase reaction is strongly pH dependent, and apparent pKa values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies, revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.

Original languageEnglish (US)
Pages (from-to)19758-19771
Number of pages14
JournalJournal of the American Chemical Society
Volume134
Issue number48
DOIs
StatePublished - Dec 5 2012

Fingerprint

dihydroneopterin aldolase
Reaction products
Mycobacterium tuberculosis
Enzymes
Fructose-Bisphosphate Aldolase
Substrates
Biosynthesis
Protons
Aldehyde-Lyases
Pterins
Folic Acid Antagonists
Racemases and Epimerases
Oxygenases
Purines
Schiff Bases
Substrate Specificity
Isotopes
Thymidine
Plasticity
Amino acids

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

One substrate, five products : Reactions catalyzed by the dihydroneopterin aldolase from Mycobacterium tuberculosis. / Czekster, Clarissa M.; Blanchard, John S.

In: Journal of the American Chemical Society, Vol. 134, No. 48, 05.12.2012, p. 19758-19771.

Research output: Contribution to journalArticle

@article{69abfb90d3c04774b8885c7b5c03f2d9,
title = "One substrate, five products: Reactions catalyzed by the dihydroneopterin aldolase from Mycobacterium tuberculosis",
abstract = "Tetrahydrofolate cofactors are required for one carbon transfer reaction involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form a protein-mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild-type DHNAs. The enzyme-substrate complex partitions 51{\%} in the first turnover to form the aldolase products, 24{\%} to the epimerase product and 25{\%} to the oxygenase products. The aldolase reaction is strongly pH dependent, and apparent pKa values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies, revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.",
author = "Czekster, {Clarissa M.} and Blanchard, {John S.}",
year = "2012",
month = "12",
day = "5",
doi = "10.1021/ja308350f",
language = "English (US)",
volume = "134",
pages = "19758--19771",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "48",

}

TY - JOUR

T1 - One substrate, five products

T2 - Reactions catalyzed by the dihydroneopterin aldolase from Mycobacterium tuberculosis

AU - Czekster, Clarissa M.

AU - Blanchard, John S.

PY - 2012/12/5

Y1 - 2012/12/5

N2 - Tetrahydrofolate cofactors are required for one carbon transfer reaction involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form a protein-mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild-type DHNAs. The enzyme-substrate complex partitions 51% in the first turnover to form the aldolase products, 24% to the epimerase product and 25% to the oxygenase products. The aldolase reaction is strongly pH dependent, and apparent pKa values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies, revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.

AB - Tetrahydrofolate cofactors are required for one carbon transfer reaction involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form a protein-mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild-type DHNAs. The enzyme-substrate complex partitions 51% in the first turnover to form the aldolase products, 24% to the epimerase product and 25% to the oxygenase products. The aldolase reaction is strongly pH dependent, and apparent pKa values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies, revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.

UR - http://www.scopus.com/inward/record.url?scp=84870667585&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84870667585&partnerID=8YFLogxK

U2 - 10.1021/ja308350f

DO - 10.1021/ja308350f

M3 - Article

C2 - 23150985

AN - SCOPUS:84870667585

VL - 134

SP - 19758

EP - 19771

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 48

ER -