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Structure-function relationships of CldA: a unique functional intermediate between starch hydrolases and
Beatriz Velazquez-Cruz1,2, Montserrat Romero-Jiménez1, Yasel Guerra3,4
1Laboratorio de Biología Sintética, Estructural y Molecular, Facultad de Ingeniería Mecánica y Eléctrica, Universidad de Colima, Mexico.
The FEBS Journal
|August 12, 2026
Summary
The CldA enzyme uniquely combines starch hydrolysis and cyclodextrin formation capabilities. Structural analysis reveals evolutionary adaptations enabling dual function, shifting specificity towards hydrolysis at high starch concentrations.
Area of Science:
- Enzymology
- Structural Biology
- Biochemistry
Background:
- Cyclomaltodextrin glucanotransferases (CGTases) are key enzymes in carbohydrate processing.
- Family 13 of glycoside hydrolases (GH13) includes enzymes with diverse starch-modifying activities.
- Understanding enzyme evolution and function is crucial for biocatalysis.
Purpose of the Study:
- To elucidate the structure and function of the CldA enzyme, an intermediate between starch hydrolases and CGTases.
- To investigate the evolutionary adaptations contributing to CldA's dual hydrolytic and transglycosylation activities.
- To provide structural insights into the coexistence of hydrolysis and cyclization in a single enzyme active site.
Main Methods:
- X-ray crystallography at 1.66 Å resolution to determine CldA structure.
- Kinetic studies to analyze enzyme activity and specificity.
- Site-directed mutagenesis and chimera construction (CldA-DE_CBM20) to probe functional mechanisms.
- Structural comparisons with related enzymes (GH13_1 and GH13_2).
Main Results:
- CldA exhibits a three-domain ABC architecture with three calcium-dependent folding centers for thermostability.
- Expanded active site cleft (-7 to +2 subsites) accommodates dual functionality.
- Key adaptations include the absence of DE_CBM20 domains, a stabilizing hydrophobic pair (Trp204/Met281), and a specific hydrogen bond (Ser200-Phe216).
- Mutant and chimera studies highlight the boundary between hydrolytic and cyclization activities.
Conclusions:
- CldA represents the first experimentally characterized native GH13_2 enzyme with coexisting hydrolytic and cyclization activities.
- Its structure reveals evolutionary divergence from canonical CGTases, lacking C-terminal domains.
- Product-length specificity shifts towards hydrolysis dependent on starch concentration, mediated by active site architecture.
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