4MZU

Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/N-Acetyltransferase from Shewanella denitrificans.

(2013) Biochemistry 52: 8374-8385

• PubMed: 24128043 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1021/bi401170t
• Primary Citation of Related Structures:  
  4MZU


• PubMed Abstract: 
  

  Unusual N-acetylated sugars have been observed on the O-antigens of some Gram-negative bacteria and on the S-layers of both Gram-positive and Gram-negative bacteria. One such sugar is 3-acetamido-3,6-dideoxy-α-d-galactose or Fuc3NAc. The pathway for its production requires five enzymes with the first step involving the attachment of dTMP to glucose-1-phosphate. Here, we report a structural and biochemical characterization of a bifunctional enzyme from Shewanella denitificans thought to be involved in the biosynthesis of dTDP-Fuc3NAc. On the basis of a bioinformatics analysis, the enzyme, hereafter referred to as FdtD, has been postulated to catalyze the third and fifth steps in the pathway, namely, a 3,4-keto isomerization and an N-acetyltransferase reaction. For the X-ray analysis reported here, the enzyme was crystallized in the presence of dTDP and CoA. The crystal structure shows that FdtD adopts a hexameric quaternary structure with 322 symmetry. Each subunit of the hexamer folds into two distinct domains connected by a flexible loop. The N-terminal domain adopts a left-handed β-helix motif and is responsible for the N-acetylation reaction. The C-terminal domain folds into an antiparallel flattened β-barrel that harbors the active site responsible for the isomerization reaction. Biochemical assays verify the two proposed catalytic activities of the enzyme and reveal that the 3,4-keto isomerization event leads to the inversion of configuration about the hexose C-4' carbon.

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Organizational Affiliation: 

Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.