Structure-function relationships of CldA: a unique functional intermediate between starch hydrolases and cyclomaltodextrin glucanotransferases.

Abstract

The CldA enzyme is an unprecedented functional intermediate exhibiting the dual hydrolytic specificity of starch hydrolases and the intramolecular transglycosylation capacity of cyclomaltodextrin glucanotransferases (CGTases) from subfamily 2 of family 13 of glycoside hydrolases (GH13_2). Here, the crystallographic structure of CldA was determined at 1.66 Å resolution. Structural and kinetic studies revealed that the thermophilic CldA has a three-domain ABC architecture similar to that of starch hydrolases from GH13_1, and it contains three calcium-dependent folding centers (Ca+2 1-3) essential for thermostability. However, it simultaneously features nine expanded subsites (-7 to +2) defining the active site cleft of the canonical five-domain ABCDECBM20 CGTases from GH13_2. Structural comparisons revealed three evolutionary adaptations in CldA: (a) the absence of the substrate-guiding DECBM20 domains; (b) an unusual hydrophobic pair, Trp204/Met281, whose hydrophobicity was critical for stabilizing the cyclodextrin (CD) ring at the acceptor subsite +2; and (c) an unexpected hydrogen bond of 2.60 Å between Ser200 at subsite -6 and the key central aromatic residue, Phe216, involved in starch circularization for CD formation. Characterization of two mutants, CldAM281F and CldAS200G, and a five-domain chimera, CldA-DECBM20, provided insights into the boundary between starch hydrolases and CGTases. CldA provides the first experimental structural evidence for a native GH13_2 enzyme where hydrolytic and cyclization activities coexist at a presculpted CGTase active site. Overall, structural and functional analysis of CldA showed that it diverges from canonical GH13_2 CGTases by lacking C-terminal DE domains, shifting its intramolecular transglycosylation specificity toward hydrolysis through an intriguing starch concentration-dependent product-length mechanism.