A hot desert hypolith metagenomic DNA sequence dataset was screened in-silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded a approximately 36 kDa protein (Axe1NaM1). The synthesised gene was cloned, expressed and the resulting protein, purified. NaM1 was optimally active at pH 8.5 and 30 degrees C, and functionally stable at salt concentrations up to 5 M. The specific activity and catalytic efficiency were 488.9 Umg(-1) and 3.26x10(6) M(-1)s(-1), respectively. The crystal structure of wild type NaM1 was solved at a resolution of 2.03 A and a comparison with the structures and models of more thermostable carbohydrate esterase (CE) 7 family enzymes and variants of NaM1 from a directed evolution experiment, respectively, suggest 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 Acetyl xylan esterases (AcXEs) belong to nine carbohydrate esterase families (CE 1-7, 12 and 16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterised 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 polymerase chain reaction (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 alpha/beta hydrolase enzymes.
Representative scheme of Acetyl-esterase_deacetylase structure and an image from PDBsum server
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