Citrate synthase from Thermus aquaticus: a thermostable bacterial enzyme with a five-membered inter-subunit ionic network

E N Karlsson, S J Crennell, C Higgins, S Nawaz, L Yeoh, D W Hough, M J Danson

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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 (Topt at 80°C), the thermal stability of the enzyme is high, as judged from its Tm (101°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.
Original languageEnglish
Pages (from-to)9-16
Number of pages8
JournalExtremophiles
Volume7
Issue number1
DOIs
Publication statusPublished - 2003

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Thermus
Citrate (si)-Synthase
Enzymes
Pyrococcus furiosus
Hot Temperature
Deinococcus
Enzyme Stability
Archaea
Molecular Structure
Gene Library
Amino Acid Sequence
Ions
Polymerase Chain Reaction
DNA
Genes

Cite this

Citrate synthase from Thermus aquaticus: a thermostable bacterial enzyme with a five-membered inter-subunit ionic network. / Karlsson, E N; Crennell, S J; Higgins, C; Nawaz, S; Yeoh, L; Hough, D W; Danson, M J.

In: Extremophiles, Vol. 7, No. 1, 2003, p. 9-16.

Research output: Contribution to journalArticle

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AU - Yeoh, L

AU - Hough, D W

AU - Danson, M J

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AB - 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 (Topt at 80°C), the thermal stability of the enzyme is high, as judged from its Tm (101°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.

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