Citrate synthase from Thermus aquaticus: a thermostable bacterial enzyme with a five-membered inter-subunit ionic network.
A bacterial thermostable citrate synthase has been analyzed to investigate the structural basis of its thermostability, and to compare such features with those previously identified in archaeal citrate synthases. The gene encoding the citrate synthase from Thermus aquaticus was identified from a gene library by screening with a PCR fragment amplified from genomic DNA using a primer based on the determined N-terminal amino acid sequence and a citrate synthase consensus primer. Apart from high sequence similarities with citrate synthase sequences within the Thermus/ Deinococcus group, the analyzed enzyme has highest similarities with the enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The recombinant enzyme is a dimer with high specific activity. Compared to its thermoactivity (T(opt)at 80 degrees C), the thermal stability of the enzyme is high, as judged from its T(m) (101 degrees C), and from irreversible thermal inactivation assays. Molecular modeling of the structure revealed an inter-subunit ion-pair network, comparable in size to the network found in the citrate synthase from P. furiosus; these networks are discussed in relation to the high thermal stability of these bacterial and archaeal enzymes.
Nordberg Karlsson
Eva
E
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden.
Crennell
Susan J
SJ
Higgins
Catherine
C
Nawaz
Salina
S
Yeoh
Louisa
L
Hough
David W
DW
Danson
Michael J
MJ
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D021122:Protein Subunits; D011994:Recombinant Proteins; D002950:Citrate (si)-Synthase