Purification, crystallization and structural elucidation of d-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation

Matthew W. Vetting, Jason T. Bouvier, John A. Gerlt, Steven C. Almo

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is d-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, d-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-l-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6Å resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. d-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.

Original languageEnglish (US)
Pages (from-to)36-41
Number of pages6
JournalActa Crystallographica Section:F Structural Biology Communications
Volume72
DOIs
StatePublished - 2016

Fingerprint

Agrobacterium tumefaciens
Lactones
Crystallization
purification
Purification
Catalytic Domain
crystallization
degradation
Degradation
mandelate racemase
Protons
Carbon
Sugar Acids
acids
protons
Phosphopyruvate Hydratase
polysaccharides
carbon
rings
biomass

Keywords

  • Agrobacterium tumefaciens
  • d-galactaro-1,4-lactone cycloisomerase
  • d-galacturonate
  • pectin

ASJC Scopus subject areas

  • Biophysics
  • Genetics
  • Structural Biology
  • Condensed Matter Physics
  • Biochemistry

Cite this

@article{132b49b935854ea38bcd5e7d8bc08c69,
title = "Purification, crystallization and structural elucidation of d-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation",
abstract = "Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is d-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, d-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-l-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6{\AA} resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. d-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.",
keywords = "Agrobacterium tumefaciens, d-galactaro-1,4-lactone cycloisomerase, d-galacturonate, pectin",
author = "Vetting, {Matthew W.} and Bouvier, {Jason T.} and Gerlt, {John A.} and Almo, {Steven C.}",
year = "2016",
doi = "10.1107/S2053230X15023286",
language = "English (US)",
volume = "72",
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journal = "Acta Crystallographica Section F:Structural Biology Communications",
issn = "1744-3091",
publisher = "John Wiley and Sons Ltd",

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T1 - Purification, crystallization and structural elucidation of d-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation

AU - Vetting, Matthew W.

AU - Bouvier, Jason T.

AU - Gerlt, John A.

AU - Almo, Steven C.

PY - 2016

Y1 - 2016

N2 - Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is d-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, d-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-l-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6Å resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. d-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.

AB - Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is d-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, d-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-l-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6Å resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. d-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.

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