Crystal structures of two monomeric triosephosphate isomerase variants identified via a directed-evolution protocol selecting for L-arabinose isomerase activity.
The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using an Escherichia coli L-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of the Escherichia coli selection strain in medium supplemented with 40 mM L-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.
Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstrasse 76, ACK 24, Berlin, Germany.
Mirja
M
Krause
Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, FIN-90014 Oulu, Finland.
Tiila Riikka
TR
Kiema
Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstrasse 76, ACK 24, Berlin, Germany.
Peter
P
Neubauer
Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, FIN-90014 Oulu, Finland.
Rik K
RK
Wierenga
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D011487:Protein Conformation; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics
D019747:Aldose-Ketose Isomerases; D014305:Triose-Phosphate Isomerase; C019715:L-arabinose isomerase
TIM barrel; enzyme engineering; non-natural enzymes; structure-based rational design; triosephosphate isomerase