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Structural and biochemical interrogation on transketolase from Pichia stipitis for new functionality
Li-Jen Hsu1,2, Ning-Shian Hsu1, Yung-Lin Wang1
1Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
Protein Engineering, Design & Selection : PEDS
|September 1, 2016
Summary
Structural insights into transketolase from Pichia stipitis (TKps) reveal key residues for enzyme function. Mutating His27 alters substrate preference, shifting reactivity to facilitate xylose assimilation.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Transketolase (TKps) from Pichia stipitis is crucial for industrial applications.
- Understanding TKps structure is key to optimizing its reaction mechanisms, substrate selectivity, and directionality.
Purpose of the Study:
- To determine the crystal structures of wild-type (WT) and mutant TKps in complex with physiological ligands.
- To elucidate the roles of specific amino acid residues in coenzyme binding, substrate recognition, and catalytic activity.
Main Methods:
- X-ray crystallography to obtain high-resolution (1.03–1.6 Å) structures of TKps complexes.
- Biochemical assays and mutagenic analysis to investigate enzyme kinetics and residue function.
Main Results:
- Seven crystal structures of WT and mutant TKps were determined.
- Residues His27, His66, His100, His261, His478, Asp473, Arg356, and Arg525 were identified as critical for coenzyme and substrate interactions.
- Mutation H27A shifted enzyme activity from backward to forward reaction preference, enhancing net xylose assimilation.
Conclusions:
- Specific residues play defined roles in thiamine diphosphate binding and substrate gating.
- His27 is particularly important for distinguishing between sedoheptulose-7-phosphate and xylulose-5-phosphate binding.
- Targeted mutagenesis of TKps can redirect its catalytic function for improved industrial bioprocesses, such as xylose assimilation.
