Lipase engineering database: Understanding and exploiting sequence–structure–function relationships

Abstract

The Lipase Engineering Database is a WWW-accessible resource on sequence–structure–function relationships of microbial lipases. Sequences of 92 microbial lipases and homologous serine hydrolases were assigned to 32 homologous families and 15 superfamilies. Multisequence alignments of all homologous families and superfamilies are provided. Functionally relevant amino acids are annotated. The catalytic serine is part of the conserved nucleophilic elbow and was identified in all sequences by its conserved signature GxSxG. The complete catalytic machinery (catalytic triad and two oxyanion hole residues) could be annotated in 91% of LED sequence entries. Published mutants and their properties are provided. The X-ray structures of 22 lipases were superposed and consistently annotated. Sequence and structure data were applied to study the role of the first oxyanion hole residues. Although the backbone amides contribute to the oxyanion hole rather than side chains, the residues are well conserved. Two sequence signatures including the first oxyanion hole residue were identified: G X and GG GX. In the G X type, the position of the first oxyanion hole residue X is stabilized by one or several anchor residues. If X is hydrophilic, it is hydrogen bonded to hydrophilic anchor residues, while hydrophobic oxyanion hole residues bind to hydrophobic pockets. The GG GX type includes short chain length specific lipases and carboxylesterases. The first oxyanion hole residue G is stabilized by interaction of the dipeptide GX with the side chain of the second oxyanion hole residues, which is a conserved alanine as C-terminal neighbour of the catalytic serine. Thus, short chain specific lipases and carboxylesterase can be identified by combining the signatures GGGX and GxSAG. Consistently annotated aligned sequences and superimposed structures of microbial lipases help to understand the functional role of individual amino acids and thus the LED is a useful tool for protein engineering. The Lipase Engineering Database is available at http://www.led.uni-stuttgart.de/.