2VEI

Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties

Summary for 2VEI

• Entry DOI: 10.2210/pdb2vei/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 2VEK 2VEL 2VEM 2VEN 3TIM 4TIM 5TIM 6TIM
• Descriptor: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE, SULFATE ION (3 entities in total)
• Functional Keywords: isomerase, triosephosphate isomerase, tim barrel, glycolysis, engineering, pentose shunt, binding pocket, gluconeogenesis, lipid synthesis, substrate specificity, fatty acid biosynthesis, tim, enzyme, monomeric, glycosome
• Biological source: TRYPANOSOMA BRUCEI BRUCEI
• Cellular location: Glycosome: P04789
• Total number of polymer chains: 3
• Total formula weight: 78599.03

Authors

Alahuhta, M.,Salin, M.,Casteleijn, M.G.,Kemmer, C.,El-Sayed, I.,Augustyns, K.,Neubauer, P.,Wierenga, R.K. (deposition date: 2007-10-24, release date: 2008-02-19, Last modification date: 2023-12-13)

Primary citation

Alahuhta, M.,Salin, M.,Casteleijn, M.G.,Kemmer, C.,El-Sayed, I.,Augustyns, K.,Neubauer, P.,Wierenga, R.K.
Structure-Based Protein Engineering Efforts with a Monomeric Tim Variant: The Importance of a Single Point Mutation for Generating an Active Site with Suitable Binding Properties.
Protein Eng.Des.Sel., 21:257-, 2008

PubMed Abstract: A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove.

PubMed: 18239072
DOI: 10.1093/PROTEIN/GZN002

Experimental method

X-RAY DIFFRACTION (1.89 Å)