CLONING, EXPRESSION AND STRUCTURE DETERMINATION OF HUMAN TRANSKETOLASE
S. Thorell, Y. Lindqvist, G. Schneider
Karolinska Institutet, division of Molecular
Structural Biology, Doktorsringen 9A1, S-171 77 Stockholm, Sweden
Transketolase is an important enzyme for most living
organisms. In photosynthetic organisms, it is a part of the
Calvin cycle and for other organisms it has an important function
in the sugar metabolism. Transketolase catalyzes a ketol transfer
from a keto sugar phosphate to an aldo sugar phosphate extending
this with a two carbon unit. The substrates used are
intermediates in glycolysis and the pentose phosphate pathway.
Transketolase functions as, together with transaldolase, a
reversible link between these pathways, corresponding to the
metabolic needs of the cell.
The functional unit of transketolase is a dimer in complex with the cofactor thiamine diphosphate ThDP and a Ca2+ ion [1]. ThDP is the biologically active derivative of vitamin B1. Defects in thiamine dependent enzymes, including transketolase, have been postulated to be involved in Wernicke Korsakoff syndrome [2-4].
The transketolase gene family includes approximately 20 known sequences with an over all identity of about 50%. There are many conserved residues known to be important for the catalysis. Sequence alignment shows that it can be divided into two subclasses. One class consists of the mammalian enzymes while the other includes species of lower eukaryotes and prokaryotes.
Within the transketolase gene family there is only one known 3D structure, the S.cerevisiae transketolase [1]. Determing the structure of human transketolase would reveal in what way the 3D structure is influenced by the sequence differences between mammalian and other transketolases. The structure determination will be made with molecular replacement, using the structure of S.cerevisiae transketolase as a model.
A gene encoding human transketolase (humtkl) [2] has been amplified with PCR using an E.coli expression vector (provided by Dr. Singleton, USA) as template. The product was cloned into pCW2, an URA3 LEU2-d 2-mm plasmid [5], for expression in S.cerevisiae. A yeast strain, H402, deficient in transketolase was transformed with the pCW2-humtkl plasmid. The protein was expressed in media lacking leucine. Expression was confirmed with an SDS-PAGE gel and activity measurements.