(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > environmental samples: NE > unidentified prokaryotic organism: NE
Molecular evidence
Database
No mutation 1 structure: 6EHN: Structure of a CE15 esterase from the marine bacterial metagenome No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MQRTCVLIVLIVTSTMWTPDPDVYAQPRGFNYDEAQVPKYTLPDPLVMVD GTKVTSAKQWNDKRRDEVQQLFEAYMYGKVPDGETELIFTDAKGERALGG AAIRKQVKISFGEKEDAPAMDLLIYLPADAKVRVPVFLGLNFHGNHTIHK DKEIWLTESWVRTNKKFGITKNKANELSRGVAAGRWQIEKAIAKGYGVAT IYCGDIDPDFNFPSNGIQAYYYKKDQTIPEKGQWGTIAAWAFGLSCAMDY FETDTDIDHKKVAVLGHSRLGKTSLWAGAIDTRFALTISNCSGCGGAALS RRRFGETVRRINTSFPHWFCSRFHQYNDKEDKLPIDQHMLIALCAPRPVL INSATEDKWADPHGEFLAAQGADAVYRMLGTGGLDAKKWPEPNKLVKSTI GYHLRPGKHDVTARDWDVYIEFADHHMTGGAE
The family 15 carbohydrate esterase (CE15) MZ0003, which derives from a marine Arctic metagenome, has a broader substrate scope than other members of this family. Here we report the crystal structure of MZ0003, which reveals that residues comprising the catalytic triad differ from previously-characterized fungal homologs, and resolves three large loop regions that are unique to this bacterial sub-clade. The catalytic triad of the bacterial CE15, which includes Asp 332 as its third member, closely resembles that of family 1 carbohydrate esterases (CE1), despite the overall lower structural similarity with members of this family. Two of the three loop regions form a subdomain that deepens the active site pocket and includes several basic residues that contribute to the high positive charge surrounding the active site. Docking simulations predict specific interactions with the sugar moiety of glucuronic-acid substrates, and with aromatically-substituted derivatives that serve as model compounds for the lignin-carbohydrate complex of plant cell walls. Molecular dynamics simulations indicate considerable flexibility of the sub-domain in the substrate-bound form, suggesting plasticity to accommodate different substrates is possible. The findings from this first reported structure of a bacterial member of the CE15 family provide insight into the basis of its broader substrate specificity.
Title: Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome De Santi C, Willassen NP, Williamson A Ref: PLoS ONE, 11:e0159345, 2016 : PubMed
BACKGROUND: The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. RESULTS: MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.
marine Arctic metagenome Carbohydrate esterase MZ0003 CE15
