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Unliganded Acetate Kinase from Thermatoga Maritima
Author(s) -
Mukhopadhyay Soma,
Hasson Miriam Sarah,
Sanders David A
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.20.5.lb59-a
Subject(s) - acetate kinase , chemistry , size exclusion chromatography , carboxylate , enzyme , thermophile , monomer , affinity chromatography , kinase , crystallography , biochemistry , escherichia coli , organic chemistry , polymer , gene
Acetate kinase (AK) catalyses the reversible transfer of the phosphate group from ATP to one of the oxygen atoms of the carboxylate group in acetate to form acetyl phosphate. Acetate kinase a dimeric protein from the thermophilic organism, Thermatoga maritima, was cloned and expressed in E. coli. AK was purified in three steps using anion exchange, hydrophobic, and gel filtration chromatography. It crystallized only at 50°C. The crystals diffracted poorly, had high mosaicity as they were subjected to temperature shock between looping out and freezing. To obtain better quality crystals at lower temperatures the purification procedure was modified to include a Heparin affinity step along with anion exchange and hydrophobic chromatography steps. Crystals from this preparation grew at 20°C. A crystal grown in 36% MPD, 100mM CHES‐9.5 and 50mM CaCl2 diffracted to 3.3Å. A data set with 97.9% completeness was solved by molecular replacement using structure (1G99) of AK from Methanosarcina Thermophila, a member of the ASKHA (acetate and sugar kinases/Hsc70/actin) family. Ten monomers were found in the asymmetric unit and crystal structure was refined to R‐factor and R‐free of 19.7% and 24.3%, respectively. Each monomer in the apo enzyme contained two domains. The two active site histidines (H122 and H179) found in the ASKHA family of enzymes were located 10Å apart compared to 6Å found in the binary complex in presence of ADP in 1G99. Further, the two domains in the apo enzyme appeared to be farther apart than what was reported to be found in the binary complexes suggesting that a major conformational change occurs when nucleotides bind to the apo‐enzyme. This research was supported by NIH grant RO1‐GM57056 and NSF CAREER award 99‐84919‐MCB.