The active site of oligogalacturonate lyase provides unique insights into cytoplasmic oligogalacturonate beta-elimination.
Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å. The model contains a Mn(2+) atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the α-proton in the -1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases.
Abbott
D Wade
DW
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA. wabbott@ccrc.uga.edu
Gilbert
Harry J
HJ
Boraston
Alisdair B
AB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D010368:Pectins / Q000737:chemistry; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015008:Yersinia enterocolitica / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D002241:Carbohydrates; D011994:Recombinant Proteins; D008345:Manganese; D010368:Pectins; D011133:Polysaccharide-Lyases; C045225:oligogalacturonate lyase