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Structures of alkaloid biosynthetic glucosidases decode substrate specificity
Liqun Xia1, Martin Ruppert, Meitian Wang
1Institute of Materia Medica, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, PR China.
ACS Chemical Biology
|October 19, 2011
Summary
Two O-glucosidases in Rauvolfia evolved distinct substrate specificities. Structural analysis reveals RG
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- The Rauvolfia alkaloid biosynthetic network contains two O-glucosidases, raucaffricine glucosidase (RG) and strictosidine glucosidase (SG).
- These enzymes catalyze distinct reactions, with RG operating downstream and SG operating upstream in the ajmaline pathway.
- RG can process strictosidine, the substrate for SG, but SG cannot process raucaffricine, the substrate for RG.
Purpose of the Study:
- To elucidate the structural basis for the differing substrate specificities of RG and SG.
- To understand the catalytic mechanisms and evolutionary divergence of these related O-glucosidases.
Main Methods:
- X-ray crystallography of RG, an inactive RG-E186Q mutant, and their complexes with ligands.
- Site-directed mutagenesis and kinetic analyses of RG and SG.
- Comparative structural analysis of RG and SG active sites.
Main Results:
- Crystal structures reveal RG possesses a "wider gate" catalytic site entrance, allowing access for strictosidine.
- SG has a "slot-like" entrance that restricts access to smaller substrates like raucaffricine.
- Specific residues, including Trp392 in RG and Trp388 in SG, along with Ser390, dictate the active site gate shape and substrate acceptance.
Conclusions:
- The distinct active site architectures explain the observed substrate specificities of RG and SG.
- Structural differences provide insights into the evolution of enzyme function and O-glucosidase chemistry.
- This study offers a molecular explanation for how similar enzymes diverge in function within a single species.
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