2Y70

CRYSTALLOGRAPHIC STRUCTURE OF GM23, MUTANT G89D, AN EXAMPLE OF CATALYTIC MIGRATION FROM TIM TO THIAMIN PHOSPHATE SYNTHASE.

Summary for 2Y70

• Entry DOI: 10.2210/pdb2y70/pdb
• Related: 1AG1 1DKW 1IIG 1IIH 1KV5 1ML1 1MSS 1MTM 1TPD 1TPE 1TPF 1TRD 1TRI 1TSI 1TTI 1TTJ 2J24 2J27 2V0T 2V2C 2V2D 2V2H 2V5L 2VEI 2VEK 2VEL 2VEM 2VEN 2WSQ 2WSR 2X16 2X1R 2X1S 2X1T 2X1U 2X2G 2Y6Z 3TIM 4TIM 5TIM 6TIM
• Descriptor: TRIOSE-PHOSPHATE ISOMERASE, SULFATE ION, ACETATE ION, ... (4 entities in total)
• Functional Keywords: isomerase
• Biological source: TRYPANOSOMA BRUCEI BRUCEI
• Cellular location: Glycosome: P04789
• Total number of polymer chains: 4
• Total formula weight: 106892.29

Authors

Saab-Rincon, G.,Olvera, L.,Olvera, M.,Rudino-Pinera, E.,Soberon, X.,Morett, E. (deposition date: 2011-01-27, release date: 2011-12-07, Last modification date: 2023-12-20)

Primary citation

Saab-Rincon, G.,Olvera, L.,Olvera, M.,Rudino-Pinera, E.,Benites, E.,Soberon, X.,Morett, E.
Evolutionary Walk between (Beta/Alpha)(8) Barrels: Catalytic Migration from Triosephosphate Isomerase to Thiamin Phosphate Synthase.
J.Mol.Biol., 416:255-, 2012

PubMed Abstract: The functionally versatile (β/α)(8) barrel scaffold was used to migrate triosephosphate isomerase (TPI) to thiamin phosphate synthase (TPS) activity, two enzymes that share the same fold but catalyze unrelated reactions through different mechanisms. The high sensitivity of the selection methodology was determinant to succeed in finding proteins with the desired activity. A combination of rational design and random mutagenesis was used to achieve the desired catalytic migration. One of the parallel directed evolution strategies followed resulted in TPI derivatives able to complement the thiamin phosphate auxotrophy phenotype of an Escherichia coli strain deleted of thiE, the gene that codes for TPS. Successive rounds of directed evolution resulted in better complementing TPI variants. Biochemical characterization of some of the evolved TPI clones demonstrated that the K(m) for the TPS substrates was similar to that of the native TPS; however and in agreement with the very slow complementation phenotype, the k(cat) was 4 orders of magnitude lower, indicating that substrate binding played a major role on selection. Interestingly, the crystal structure of the most proficient variant showed a slightly modified TPI active site occupied by a thiamin-phosphate-like molecule. Substitution of key residues in this region reduced TPS activity, strongly suggesting that this is also the catalytic site for the evolved TPS activity. The presence of the TPS reaction product at the active site explains the fast inactivation of the enzyme observed. In conclusion, by combining rational design, random mutagenesis and a very sensitive selection, it is possible to achieve enzymatic activity migration.

PubMed: 22226942
DOI: 10.1016/J.JMB.2011.12.042

Experimental method

X-RAY DIFFRACTION (2.3 Å)