TY - JOUR
T1 - Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
AU - Salin, Mikko
AU - Kapetaniou, Evangelia G.
AU - Vaismaa, Matti
AU - Lajunen, Marja
AU - Casteleijn, Marco G.
AU - Neubauer, Peter
AU - Salmon, Laurent
AU - Wierenga, Rik K.
PY - 2010/8/1
Y1 - 2010/8/1
N2 - Crystallographic binding studies have been carried out to probe the active-site binding properties of a monomeric variant (A-TIM) of triosephosphate isomerase (TIM). These binding studies are part of a structure-based directed-evolution project aimed towards changing the substrate specificity of monomeric TIM and are therefore aimed at finding binders which are substrate-like molecules. A-TIM has a modified more extended binding pocket between loop-7 and loop-8 compared with wild-type TIM. The A-TIM crystals were grown in the presence of citrate, which is bound in the active site of each of the two molecules in the asymmetric unit. In this complex, the active-site loops loop-6 and loop-7 adopt the closed conformation, similar to that observed in liganded wild-type TIM. Extensive crystal-soaking protocols have been developed to flush the bound citrate out of the active-site pocket of both molecules and the crystal structure shows that the unliganded open conformation of the A-TIM active site is the same as in unliganded wild-type TIM. It is also shown that sulfonate compounds corresponding to the transition-state analogue 2-phosphoglycolate bind in the active site, which has a closed conformation. It is also shown that the new binding pocket of A-TIM can bind 3-phosphoglycerate (3PGA; an analogue of a C4-sugar phosphate) and 4-phospho-d-erythronohydroxamic acid (4PEH; an analogue of a C5-sugar phosphate). Therefore, these studies have provided a rationale for starting directed-evolution experiments aimed at generating the catalytic properties of a C5-sugar phosphate isomerase on the A-TIM framework.
AB - Crystallographic binding studies have been carried out to probe the active-site binding properties of a monomeric variant (A-TIM) of triosephosphate isomerase (TIM). These binding studies are part of a structure-based directed-evolution project aimed towards changing the substrate specificity of monomeric TIM and are therefore aimed at finding binders which are substrate-like molecules. A-TIM has a modified more extended binding pocket between loop-7 and loop-8 compared with wild-type TIM. The A-TIM crystals were grown in the presence of citrate, which is bound in the active site of each of the two molecules in the asymmetric unit. In this complex, the active-site loops loop-6 and loop-7 adopt the closed conformation, similar to that observed in liganded wild-type TIM. Extensive crystal-soaking protocols have been developed to flush the bound citrate out of the active-site pocket of both molecules and the crystal structure shows that the unliganded open conformation of the A-TIM active site is the same as in unliganded wild-type TIM. It is also shown that sulfonate compounds corresponding to the transition-state analogue 2-phosphoglycolate bind in the active site, which has a closed conformation. It is also shown that the new binding pocket of A-TIM can bind 3-phosphoglycerate (3PGA; an analogue of a C4-sugar phosphate) and 4-phospho-d-erythronohydroxamic acid (4PEH; an analogue of a C5-sugar phosphate). Therefore, these studies have provided a rationale for starting directed-evolution experiments aimed at generating the catalytic properties of a C5-sugar phosphate isomerase on the A-TIM framework.
KW - enzyme engineering
KW - hydroxamate
KW - non-natural enzymes
KW - protein crystallographic binding studies
KW - rational design
KW - TIM barrel
UR - http://www.scopus.com/inward/record.url?scp=77955455227&partnerID=8YFLogxK
U2 - 10.1107/S0907444910025710
DO - 10.1107/S0907444910025710
M3 - Article
C2 - 20693693
AN - SCOPUS:77955455227
SN - 0907-4449
VL - 66
SP - 934
EP - 944
JO - Acta Crystallographica Section D: Biological Crystallography
JF - Acta Crystallographica Section D: Biological Crystallography
IS - 8
ER -