The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima

Sun Mi Shin, Jin Myung Choi, Eric Di Luccio, Yong Jik Lee, Sang Jae Lee, Sang Jun Lee, Sung Haeng Lee, Dong Woo Lee

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80°C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.

Original languageEnglish
Pages (from-to)39-51
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume585
DOIs
Publication statusPublished - 2015 Nov 1

Fingerprint

Thermotoga maritima
Eubacterium
Racemases and Epimerases
Uridine Diphosphate
Hexoses
Galactose
Uridine Diphosphate Glucose
Substrates
Metabolism
Enzymes
UDPglucose 4-Epimerase
Uridine Diphosphate Galactose
Temperature
Substrate Specificity
Eukaryota
Gene encoding
galactose epimerase
2-(3-ethylureido)-6-methylpyridine
Molecular mass
Sugars

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Shin, Sun Mi ; Choi, Jin Myung ; Di Luccio, Eric ; Lee, Yong Jik ; Lee, Sang Jae ; Lee, Sang Jun ; Lee, Sung Haeng ; Lee, Dong Woo. / The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima. In: Archives of Biochemistry and Biophysics. 2015 ; Vol. 585. pp. 39-51.
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title = "The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima",
abstract = "UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 {\AA} and 2.0 {\AA}, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80°C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.",
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The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima. / Shin, Sun Mi; Choi, Jin Myung; Di Luccio, Eric; Lee, Yong Jik; Lee, Sang Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong Woo.

In: Archives of Biochemistry and Biophysics, Vol. 585, 01.11.2015, p. 39-51.

Research output: Contribution to journalArticle

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T1 - The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima

AU - Shin, Sun Mi

AU - Choi, Jin Myung

AU - Di Luccio, Eric

AU - Lee, Yong Jik

AU - Lee, Sang Jae

AU - Lee, Sang Jun

AU - Lee, Sung Haeng

AU - Lee, Dong Woo

PY - 2015/11/1

Y1 - 2015/11/1

N2 - UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80°C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.

AB - UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80°C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.

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