7-29: Atypical active site in an esterase isolated from cow rumen

A fosmid library of over 30.000 clones was constructed from metagenomic DNA isolated from microorganisms inhabiting the rumen of Uruguayan cows.  Fragments of 40kb were purified and cloned into fosmids, packaged into phages and used to infect E.coli strain EPI300T1R.  The library was screened for the expression of lipases and esterases using glyceryl tributyrate as substrate.   Eleven positive lipases and/or esterases clones were identified with this strategy.  The genes responsible for these activities were identified by means of a Tn5-based generalized mutagenesis.  One of them termed Lip10, showed a high degree of homology with Est5S, a putative esterase from an uncultured bacteria previously found in cow rumen but lacking further characterization.  Lip10 belongs to the DUF3089 superfamily that congregates hydrolases of unknown function.  The predicted amino acidic sequence showed an altered catalytic triad.  The catalytic serine at position 187 is in a conserved GHSQG pentapeptide which is present in all the members of the family.  Similarly, the putative catalytic aspartate residue, at position 68, is followed by a Leu or Val residue as in all the members of the family. No conserved histidine is found that could play a role at the active site besides the one forming part of the pentapeptide.  Furthermore, the aspartate residue precedes the serine in the ORF sequence which is uncommon for the active sites of esterases and lipases.  The ORF of Lip10 was cloned and expressed as a 6xHis-tagged recombinant protein and is currently being characterized in terms of preferred substrates, optimal pH and temperatures.