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PDBsum entry 3dr4
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References listed in PDB file
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Key reference
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Title
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Accommodation of gdp-Linked sugars in the active site of gdp-Perosamine synthase.
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Authors
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P.D.Cook,
A.E.Carney,
H.M.Holden.
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Ref.
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Biochemistry, 2008,
47,
10685-10693.
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PubMed id
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Abstract
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Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of
several dideoxy sugars found in the O-antigens of such infamous Gram-negative
bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the
bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine.
Three enzymes are required for the biosynthesis of GDP-perosamine starting from
mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase
from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine
synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The
enzyme is PLP-dependent and belongs to the aspartate aminotransferase
superfamily. It contains the typically conserved active site lysine residue,
which forms a Schiff base with the PLP cofactor. Two crystal structures were
determined for this investigation: a site-directed mutant protein (K186A)
complexed with GDP-perosamine and the wild-type enzyme complexed with an
unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and
1.7 A resolution, respectively, revealed the manner in which products, and
presumably substrates, are accommodated within the active site pocket of
GDP-perosamine synthase. Additional kinetic analyses using both the natural and
unnatural substrates revealed that the K m for the unnatural substrate was
unperturbed relative to that of the natural substrate, but the k cat was lowered
by a factor of approximately 200. Taken together, these studies shed light on
why GDP-perosamine synthase functions as an aminotransferase whereas another
very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase
or ColD, catalyzes a dehydration reaction using the same substrate.
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