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Probing the role of the N‐terminus of Thermodesulfovibrio yellowstonii ADPglcuose pyrophosphorylase in allosteric regulation (769.9)
Author(s) -
Yik Eric,
Susoeff Michael,
Kaur Sandeep,
Dua Gautam,
Holling Jaqueline,
Meyer Christopher
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.769.9
Subject(s) - allosteric regulation , enzyme , limiting , mutagenesis , biochemistry , chemistry , enzyme kinetics , stereochemistry , gene , active site , mutation , mechanical engineering , engineering
ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. The successful engineering of ADPG PPase will allow for the production of more renewable and biodegradable carbon. Thermodesulfovibrio yellowstonii (Td.y) ADPG PPase shows only ~30% identity to other bacterial forms of the enzyme; at positions E15, F18, F23, S25, and S28 the Td.y enzyme differs from consensus in the glycine rich (positions 9‐17) and KRAKPAV regions (positions 23‐29), areas known to play roles in activity and allosteric regulation. These positions were targeted for mutagenesis and the enzymes purified using hydroxyapitite and DEAE chromatography. For wild‐type (WT), kinetic studies revealed a V max of 5.3 U/mg and S 0.5 values of 4.0 mM, 13.9 mM, and 1.2 mM for ATP, Mg, and G1P, respectively. Metabolites PEP, G6P, and 3PGA increased the V max by 1.5‐3 fold and decreased the S 0.5 values for ATP by 4.2, 10.3, and 3.8 fold, respectively. Initial kinetics for the variant S25A gave a V max ~3 fold higher and a 10‐fold lower S 0.5 value for ATP compared to WT. S25 in Td.y may play a role in setting the activity level in the absence of effectors. Further experiments are being conducted to fully characterize S25A and other variants, particularly with respect to allosteric regulation. Efforts are also underway to crystallize these enzymes to solve the three dimensional structures. Grant Funding Source : NSF Grant 0448676 and a CSUPERB Presidential Commission Grant