Mechanistic diversity in the RuBisCO superfamily: The "enolase" in the methionine salvage pathway in Geobacillus kaustophilus

Heidi J. Imker, Alexander A. Fedorov, Elena V. Fedorov, Steven C. Almo, John A. Gerlt

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ("enolization") of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5′-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this "enolase" is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO-and "enolase"-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg2+, we sought to establish structure-function relationships for the "enolase" reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the "enolases" from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the "enolase"-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated "enolase" from G. kaustophilus (carboxylated on Lys 173) liganded with Mg2+ and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal α+β domain instead of the C-terminal (β/α)8-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all "enolases", is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus "enolase" is unable to catalyze the "enolase" reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base.

Original languageEnglish (US)
Pages (from-to)4077-4089
Number of pages13
JournalBiochemistry
Volume46
Issue number13
DOIs
StatePublished - Apr 3 2007

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Geobacillus
Salvaging
Oxygenases
Phosphopyruvate Hydratase
Methionine
Protons
Ribose
Bacilli
Substrates
Bacillus
Anions
ribulose-1,5 diphosphate
Spinacia oleracea
Carbamates
Enzymes
Bacillus subtilis
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanistic diversity in the RuBisCO superfamily : The "enolase" in the methionine salvage pathway in Geobacillus kaustophilus. / Imker, Heidi J.; Fedorov, Alexander A.; Fedorov, Elena V.; Almo, Steven C.; Gerlt, John A.

In: Biochemistry, Vol. 46, No. 13, 03.04.2007, p. 4077-4089.

Research output: Contribution to journalArticle

Imker, Heidi J. ; Fedorov, Alexander A. ; Fedorov, Elena V. ; Almo, Steven C. ; Gerlt, John A. / Mechanistic diversity in the RuBisCO superfamily : The "enolase" in the methionine salvage pathway in Geobacillus kaustophilus. In: Biochemistry. 2007 ; Vol. 46, No. 13. pp. 4077-4089.
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abstract = "D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ({"}enolization{"}) of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5′-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this {"}enolase{"} is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO-and {"}enolase{"}-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg2+, we sought to establish structure-function relationships for the {"}enolase{"} reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the {"}enolases{"} from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the {"}enolase{"}-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated {"}enolase{"} from G. kaustophilus (carboxylated on Lys 173) liganded with Mg2+ and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal α+β domain instead of the C-terminal (β/α)8-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all {"}enolases{"}, is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus {"}enolase{"} is unable to catalyze the {"}enolase{"} reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base.",
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T1 - Mechanistic diversity in the RuBisCO superfamily

T2 - The "enolase" in the methionine salvage pathway in Geobacillus kaustophilus

AU - Imker, Heidi J.

AU - Fedorov, Alexander A.

AU - Fedorov, Elena V.

AU - Almo, Steven C.

AU - Gerlt, John A.

PY - 2007/4/3

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N2 - D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ("enolization") of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5′-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this "enolase" is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO-and "enolase"-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg2+, we sought to establish structure-function relationships for the "enolase" reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the "enolases" from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the "enolase"-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated "enolase" from G. kaustophilus (carboxylated on Lys 173) liganded with Mg2+ and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal α+β domain instead of the C-terminal (β/α)8-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all "enolases", is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus "enolase" is unable to catalyze the "enolase" reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base.

AB - D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ("enolization") of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5′-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this "enolase" is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO-and "enolase"-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg2+, we sought to establish structure-function relationships for the "enolase" reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the "enolases" from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the "enolase"-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated "enolase" from G. kaustophilus (carboxylated on Lys 173) liganded with Mg2+ and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal α+β domain instead of the C-terminal (β/α)8-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all "enolases", is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus "enolase" is unable to catalyze the "enolase" reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base.

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