3DR7

GDP-perosamine synthase from Caulobacter crescentus with bound GDP-3-deoxyperosamine

Summary for 3DR7

• Entry DOI: 10.2210/pdb3dr7/pdb
• Related: 3bn1 3dr4
• Descriptor: Putative perosamine synthetase, (2R,3S,5S,6R)-5-amino-3-hydroxy-6-methyl-oxan-2-yl, 1,2-ETHANEDIOL, ... (4 entities in total)
• Functional Keywords: perosamine, pyridoxal phosphate, o-antigen, lipopolysaccharide, aspartate aminotransferase, deoxysugar, transferase
• Biological source: Caulobacter crescentus
• Total number of polymer chains: 4
• Total formula weight: 175482.32

Authors

Holden, H.M.,Cook, P.D.,Carney, A.E. (deposition date: 2008-07-10, release date: 2008-10-14, Last modification date: 2023-11-15)

Primary citation

Cook, P.D.,Carney, A.E.,Holden, H.M.
Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase
Biochemistry, 47:10685-10693, 2008

PubMed Abstract: 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.

PubMed: 18795799
DOI: 10.1021/bi801309q

Experimental method

X-RAY DIFFRACTION (1.7 Å)