Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-D-galactarate Dehydratase

Helena Taberman; Martina Andberg; Tarja Parkkinen; Janne Janis; Merja Penttilä; Nina Hakulinen; Anu Koivula; Juha Rouvinen

doi:10.1021/bi501290k

Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-D-galactarate Dehydratase

Helena Taberman
, Martina Andberg
, Tarja Parkkinen
, Janne Janis
, Merja Penttilä
, Nina Hakulinen
, Anu Koivula
, Juha Rouvinen^*

^*Corresponding author for this work

Not published at Aalto University

Research output: Contribution to journal › Article › Scientific › peer-review

Abstract

Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce a-ketoglutaric semialdehyde (alpha-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 angstrom 1.5 angstrom, and 2.1 angstrom resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

Original language	English
Pages (from-to)	8052-8060
Number of pages	9
Journal	Biochemistry
Volume	53
Issue number	51
DOIs	https://doi.org/10.1021/bi501290k
Publication status	Published - 30 Dec 2014
MoE publication type	A1 Journal article-refereed

Funding

This work was supported by the National Doctoral Programme in Informational and Structural Biology, and the Academy of Finland through the Finnish Centre of Excellence in White Biotechnology-Green Chemistry (Decision 118573). The research leading to these results has received funding also from the European Community's Seventh Framework Pro-gramme (FP7/2007-2013) under BioStruct-X (grant agreement No283570).

Keywords

MACROMOLECULAR CRYSTALLOGRAPHY
ENZYMATIC-ACTIVITIES
ENOLASE SUPERFAMILY
D-GLUCARATE
DEHYDROGENASE
EVOLUTION
LYASE
PURIFICATION
REFINEMENT
MECHANISM

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10.1021/bi501290k

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title = "Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-D-galactarate Dehydratase",

abstract = "Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce a-ketoglutaric semialdehyde (alpha-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 angstrom 1.5 angstrom, and 2.1 angstrom resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.",

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author = "Helena Taberman and Martina Andberg and Tarja Parkkinen and Janne Janis and Merja Penttil{\"a} and Nina Hakulinen and Anu Koivula and Juha Rouvinen",

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doi = "10.1021/bi501290k",

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T1 - Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-D-galactarate Dehydratase

AU - Taberman, Helena

AU - Andberg, Martina

AU - Parkkinen, Tarja

AU - Janis, Janne

AU - Penttilä, Merja

AU - Hakulinen, Nina

AU - Koivula, Anu

AU - Rouvinen, Juha

PY - 2014/12/30

Y1 - 2014/12/30

N2 - Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce a-ketoglutaric semialdehyde (alpha-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 angstrom 1.5 angstrom, and 2.1 angstrom resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

AB - Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce a-ketoglutaric semialdehyde (alpha-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 angstrom 1.5 angstrom, and 2.1 angstrom resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

KW - MACROMOLECULAR CRYSTALLOGRAPHY

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KW - ENOLASE SUPERFAMILY

KW - D-GLUCARATE

KW - DEHYDROGENASE

KW - EVOLUTION

KW - LYASE

KW - PURIFICATION

KW - REFINEMENT

KW - MECHANISM

U2 - 10.1021/bi501290k

DO - 10.1021/bi501290k

M3 - Article

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JO - Biochemistry

JF - Biochemistry

IS - 51

ER -

Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-D-galactarate Dehydratase

Abstract

Funding

Keywords

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