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Characterization of the Recombinant ADPGlucose Pyrophosphorylase from Rhodopsuedomonas palustris . Role of the Extreme N‐terminus and Ser‐44
Author(s) -
Eng Chesda,
Yetemian Rosanne,
Purcell Dan,
Thurner Kristi,
Van Vo Thanh,
Nguyen Christine,
Cummings Patty,
Meyer Christopher R.
Publication year - 2006
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.20.4.a53
Subject(s) - biochemistry , enzyme , recombinant dna , gene , expression vector , pbr322 , microbiology and biotechnology , activator (genetics) , chemistry , biology
ADPGlucose Pyrophosphorylase (ADPG PPase, glgC gene product) catalyzes a key step in glucan synthesis and is allosterically regulated. Engineering of the ADPG PPase from Rhodopseudomonas palustris CGA009 ( Rps.p .) is of interest as this versatile bacteria has the capacity for carbon sequestration. The Rps.p. glgC gene was amplified from isolated genomic DNA as an NcoI‐BglII fragment and subcloned into the pSE420 (Invitrogen) expression vector. An error in the provisional annotation of the gene led to the expression and purification of an altered protein with four extra amino acids at the N‐terminus that had a specific activity of only 5.4 U/mg which was weakly activated by pyruvate, F6P, and FBP. The N‐terminus was corrected by PCR mutagenesis to match the current conceptual translation (NC_005296.1: 416649..417911) and the recombinant protein purified by a heat step and DEAE, phenyl sepharose, and UNO Q (BioRad) anion exchange chromatography to yield an enzyme with a specific activity of ~100 U/mg. The enzyme had S 0.5 values for ATP, Mg, and Glc‐1‐P in accord with other bacterial ADPG PPases and was activated by F6P and pyruvate and inhibited by FBP which serves as an activator of other ADPG PPases. Based on alignment studies, we hypothesized that S44, A378, K382, and A385 could be responsible for FBP inhibition. The purified S48A enzyme was found to have a specific activity of ~120 U/mg and was desensitized to FBP. Additional mutagenesis is underway. Supported in part by NSF grant 0448676.