Structure and Active Site Residues of PglD, an N-Acetyltransferase from the Bacillosamine Synthetic Pathway Required for N-Glycan Synthesis in Campylobacter jejuni
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- 作者：Erumbi S. Rangarajan ; Karen M. Ruane ; Traian Sulea ; David C. Watson ; Ariane Proteau ; Sonia Leclerc ; Miroslaw Cygler ; Allan Matte ; N. Martin Young
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：February 19, 2008
- 年：2008
- 卷：47
- 期：7
- 页码：1827 - 1836
- 全文大小：787K
- 年卷期：v.47,no.7(February 19, 2008)
- ISSN：1520-4995
文摘
Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. Theheptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2,4-diacetamido-2,4,6-trideoxy-D-Glc (QuiNAc4NAc or N,N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar'ssynthesis is the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-D-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms,refined to 1.8 and 1.75 Å resolution, respectively. PglD is a trimer of subunits each comprised of two domains,an N-terminal 
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-domain and a C-terminal left-handed -helix. Few structural differences accompany CoAbinding, except in the C-terminal region following the -helix (residues 189-195), which adopts an extendedstructure in the unbound form and folds to extend the -helix upon binding CoA. Computational moleculardocking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with themolecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modelingindicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' themost likely residue to function as a general base, removing H+ from the amino group prior to nucleophilicattack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed theimportance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing theintricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKaof the putative general base, His125'.
/-domain and a C-terminal left-handed -helix. Few structural differences accompany CoAbinding, except in the C-terminal region following the -helix (residues 189-195), which adopts an extendedstructure in the unbound form and folds to extend the -helix upon binding CoA. Computational moleculardocking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with themolecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modelingindicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' themost likely residue to function as a general base, removing H+ from the amino group prior to nucleophilicattack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed theimportance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing theintricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKaof the putative general base, His125'.