Structural and kinetic studies of sugar binding to galactose mutarotase from Lactococcus lactis

James B. Thoden, Jungwook Kim, Frank M. Raushel, Hazel M. Holden

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Galactose mutarotase catalyzes the conversion of β-D-galactose to α-D-galactose in the Leloir pathway for galactose metabolism. The high resolution x-ray structure of the dimeric enzyme from Lactococcus lactis was recently solved and shown to be topologically similar to the 18-stranded, anti-parallel β-motif observed for domain 5 of β-galactosidase. In addition to determining the overall molecular fold of galactose mutarotase, this initial investigation also provided a detailed description of the electrostatic interactions between the enzyme and its physiologically relevant substrate, galactose. Specifically, the side chains of His-96 and His-170 were shown to be located within hydrogen bonding distance to the C-5 oxygen of the substrate, while the carboxylate of Glu-304 was positioned near the C-1 hydroxyl group of the sugar. On the basis of this initial study, a possible role for Glu-304 as the general acid/base group in catalysis was put forth. Here we describe the combined x-ray crystallographic and kinetic analyses of L. lactis galactose mutarotase complexed with D-glucose, D-fucose, D-quinovose, L-arabinose, or D-xylose. These investigations have revealed that there are several distinct binding modes for these sugars, which are dependent upon the spatial orientation of the C-4 hydroxyl group. In those sugars with the same C-4 hydroxyl group orientation as galactose, their C-1 hydroxyl groups are invariably located near Glu-304. For those sugars, which have the same C-4 hydroxyl group configuration as glucose, the C-1 hydroxyls are typically located near Asp-243. These different binding modes correlate with both the observed kinetic parameters and the presence or absence of a hydrogen bond between the guanidinium group of Arg-71 and the C-4 hydroxyl group of the sugar ligand.

Original languageEnglish (US)
Pages (from-to)45458-45465
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number47
DOIs
StatePublished - Nov 22 2002
Externally publishedYes

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Lactococcus lactis
Sugars
Hydroxyl Radical
Galactose
Kinetics
Hydrogen bonds
X-Rays
Galactosidases
Glucose
X rays
Arabinose
Fucose
Xylose
Guanidine
Substrates
Enzymes
Hydrogen Bonding
Coulomb interactions
galactose mutarotase
Static Electricity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural and kinetic studies of sugar binding to galactose mutarotase from Lactococcus lactis. / Thoden, James B.; Kim, Jungwook; Raushel, Frank M.; Holden, Hazel M.

In: Journal of Biological Chemistry, Vol. 277, No. 47, 22.11.2002, p. 45458-45465.

Research output: Contribution to journalArticle

Thoden, James B. ; Kim, Jungwook ; Raushel, Frank M. ; Holden, Hazel M. / Structural and kinetic studies of sugar binding to galactose mutarotase from Lactococcus lactis. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 47. pp. 45458-45465.
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