Highlights• AcXEs occur in several CAZy families, and show promiscuous substrate specificities.• A strong AcXE sequence to specificity link would be a valuable functional predictor.• There is a clear need for novel AcXE enzymes with detailed substrate specificity data.• Functional metagenomics would be the most effective means for identifying new AcXEs.• A lack of suitable substrates limits high throughput metagenomic biomining of AcXEs.

A hot desert hypolith metagenomic DNA sequence data set was screened for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1). 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 and 3.26 × 10 M s, 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. 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 (Axe1), 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/β hydrolase enzymes.

No abstract

The carbohydrate esterase (CE) 7 enzymes are known for their specificity for a broad range of carboxyl ester substrates [1, 2]. Most studies investigating the basis for substrate specificity and thermal characteristics have been carried out on a highly thermostable member of this family [3, 4] and little is known about the thermolability determinants of homologous enzymes. Here we describe the crystal structure of a novel CE7 acetyl xylan esterase designated NaM1. NaM1 was encoded by a 966bp gene (NaMet1) obtained via in silico bio-mining of a Namib Desert hypolith metagenome, followed by chemical synthesis of the full length gene. Following cloning and expression, His6-tagged NaM1 was purified by cobalt-affinity and fast-pressure liquid chromatography to >95% purity. Protein crystals obtained from sitting-drop crystallization experiments yielded a 1.7 Å X-ray diffraction dataset, allowing the NaM1 structure to be solved by molecular replacement. Functional analysis revealed NaM1 to be a thermolabile enzyme with optimal activity at 35 o C (pH 8), the lowest reported for the CE7 family. NaM1 degraded p-nitrophenol acetate, p-NP butyrate, 7-aminocephalosporanic acid and acetylated xylan. The crystal structure provides a basis for comparing substrate specificities and thermolability in CE7 deacetylases.[1] Biely, Biotechnol. Adv. 2012, 30, 1575-1588 Adesioye et al., Enzyme Microb. Technol. 2016, 93, 79-91 [3] Hedge et al