Acetyl xylan esterases (AcXEs) are crucial for biomass degradation. However, the catalytic mechanism underlying the highly specific activity remains poorly understood, limiting their rational engineering. Here, we characterized the previously undescribed carbohydrate esterase family (CE) 7 acetyl xylan esterase (LaCE7A) from Lactococcus lactis with high specific activity (154 179 U·mg-1) to unravel the mechanism underlying its efficient catalysis. The monomer structure of LaCE7A presented a typical α/β-hydrolase fold and contained three distinct structure features in CE family 7, exhibiting evolutionary conservation. The large number of hydrophilic residues in the active pocket may increase the affinity of the reaction intermediates to hydrophilic substrates, thus facilitating substrate binding of LaCE7A, which may further contribute to the high specific activity observed. MD simulations indicated that the flexibilities of two regions (residues 139-144 and residues 220-223) increased the volume of the active pocket of the enzyme and were conducive to substrate binding and catalytic reaction. In addition, the unique Ala220 located in the tight turn region of three-helix insertion domain adopted the more favorable trans peptide bond, which played a crucial role in substrate catalysis. Our findings not only identify LaCE7A as a potent biocatalyst but also provide new mechanistic insights into the high activity of CE7 AcXEs, offering a foundation for future enzyme design.
Lv et al. (Fri,) studied this question.