4PC8

Structure-based protein engineering efforts on the scaffold of a monomeric triosephosphate isomerase yielding a sugar isomerase

Summary for 4PC8

• Entry DOI: 10.2210/pdb4pc8/pdb
• Related: 1AG1 1DKW 1IIG 1ML1 1MSS 1MTM 1TPD 1TPE 1TRI 2VEK 2VEL 5TIM
• Descriptor: Ma21-TIM, GLYCOLIC ACID (3 entities in total)
• Functional Keywords: triosephosphate isomerase tim barrel protein engineering substrate specificity, isomerase
• Biological source: Trypanosoma brucei brucei
• Total number of polymer chains: 1
• Total formula weight: 25851.56

Authors

Krause, M.,Neubauer, P.,Wierenga, R.K. (deposition date: 2014-04-14, release date: 2015-04-22, Last modification date: 2023-11-15)

Primary citation

Krause, M.,Kiema, T.R.,Neubauer, P.,Wierenga, R.K.
Crystal structures of two monomeric triosephosphate isomerase variants identified via a directed-evolution protocol selecting for L-arabinose isomerase activity.
Acta Crystallogr.,Sect.F, 72:490-499, 2016

PubMed Abstract: 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.

PubMed: 27303904
DOI: 10.1107/S2053230X16007548

Experimental method

X-RAY DIFFRACTION (1.55 Å)