Structural Characterization and Directed Evolution of a Novel Acetyl Xylan Esterase Reveals Thermostability Determinants of the Carbohydrate Esterase 7 Family.

A hot desert hypolith metagenomic DNA sequence data set was screened in silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1NaM1). The synthesized gene was cloned and expressed, and the resulting protein was purified. NaM1 was optimally active at pH 8.5 and 30°C and functionally stable at salt concentrations of up to 5 M. The specific activity and catalytic efficiency were 488.9 U mg-1 and 3.26 × 106 M-1 s-1, respectively. The crystal structure of wild-type NaM1 was solved at a resolution of 2.03 Å, and a comparison with the structures and models of more thermostable carbohydrate esterase 7 (CE7) family enzymes and variants of NaM1 from a directed evolution experiment suggests that reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1. Surprisingly, a single point mutation (N96S) not only resulted in a simultaneous improvement in thermal stability and catalytic efficiency but also increased the acyl moiety substrate range of NaM1.IMPORTANCE AcXEs belong to nine carbohydrate esterase families (CE1 to CE7, CE12, and CE16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterized members of this family are moderately to highly thermostable. The crystal structure of a novel, mesophilic CE7 AcXE (Axe1NaM1), from a soil metagenome, provides a basis for comparisons with thermostable CE7 enzymes. Using error-prone PCR and site-directed mutagenesis, we enhanced both the stability and activity of the mesophilic AcXE. With comparative structural analyses, we have also identified possible thermal stability determinants. These are valuable for understanding the thermal stability of enzymes within this family and as a guide for future protein engineering of CE7 and other α/ß hydrolase enzymes.

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.

Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products. Although one-third of all characterized CEs within CAZy families 1-7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks. The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.