Determination of catalytic and substrate-interaction residues in a thermostable pectate lyase
Monday, April 28, 2014
Exhibit/Poster Hall, lower level (Hilton Clearwater Beach)
Markus Alahuhta, Larry E. Taylor, Roman Brunecky, Michael E. Himmel and Vladimir V. Lunin, Biosciences Center, National Renewable Energy Laboratory, Golden, CO
We recently acquired and solved the X-ray structure of the catalytic domain of the family 3 pectate lyase (PL3-cat) from the cellulolytic thermophile Caldicellulosiruptor bescii. In a follow up study we obtained a structure which included the products of the trigalacturonic acid in the active site (PDB 4ew9); analysis of this structure and comparisons to other PL3 and PL1 structures, revealed that: 1) the active site architecture of PL3 enzymes differ significantly from PL1, 2) Glu84 appears to be the catalytic acid and 3) Lys108 is most likely the catalytic base which acts by abstracting a proton from the C5 of the Glucuronic acid residue at the -1 subsite. As lysine has not been widely known to play this role in carbohydrate active enzymes, we performed a series of mutational studies to definitively identify the catalytic residues. Activity assays confirmed the proposed functions of Glu84 and Lys108.