Rhodococcus sp. strain CR-53 LipR, the first member of a new bacterial lipase family (family X) displaying an unusual Y-type oxyanion hole, similar to the Candida antarctica lipase clan

Abstract

Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.

Fig 1

Selected aspects of LipR characterization assayed on pNP-caprate. (A) Optimum temperature; (B) optimum pH; (C) effect of several ions on LipR activity. The activity values are the means of at least three independent determinations.

Fig 2

3D homology model of LipR with the catalytic residues, the conserved motifs, the disulfide bond, and the oxyanion hole highlighted. (A) The amino acids of the conserved blocks among family X bacterial lipases are mostly located on LipR in the region of the active site and are depicted here as blue spheres. Among them, the amino acids forming the consensus motif of the oxyanion hole are depicted as orange spheres. The amino acids forming the catalytic triad are red sticks, and the disulfide bond formed by the two cysteines (Cys³⁸⁸ and Cys⁴³²) is shown as yellow sticks. (B) Detail showing the position of the conserved motif Tyr-Asp-Ser-Leu (as spheres) constituting the Y-type oxyanion hole of LipR, which has never been found before among bacterial lipases but which is shared with most lipases of the C. antarctica clan. The two proposed amino acids involved in the oxyanion hole (Tyr¹¹⁰ and Asp¹¹¹) are depicted as orange sticks, and the catalytic serine (Ser²¹²) and histidine (His⁴⁰⁴) are depicted as red sticks. The 3D model was obtained through YASARA.

Fig 3

Phylogenetic tree including the amino acid sequences of putative, uncharacterized lipases showing over 30% identity to LipR. The blue box includes lipases of the LipR cluster. Green box, cluster constituted by putative lipases from Mycobacterium species; orange box, cluster including putative lipases from different Rhodococcus species. The tree was obtained using the software MEGA4 (44).

Fig 4

Blocks of sequences conserved in the LipR cluster of putative bacterial lipases assigned to bacterial lipase family X. The aligned sequences were the putative lipases forming the LipR cluster. YP_002769179, putative lipase from R. erythropolis PR4; ZP_04383612, putative triacylglycerol lipase from R. erythropolis SK121; YP_345621, putative lipase from R. erythropolis PR4; ZP_04386725, triacylglycerol lipase from R. erythropolis SK121; ZP_07281779, putative triacylglycerol lipase from Streptomyces sp. strain AA4; ZP_06908675, putative triacylglycerol lipase from S. pristinaespiralis ATCC 25486; ZP_04693904, putative lipase from S. roseosporus NRRL 15998; YP_003111889, putative triacylglycerol lipase from Catenulispora acidiphila DSM 44928; ZP_07716111, putative triacylglycerol lipase from Aeromicrobium marinum DSM 15272; YP_002235365, putative exported lipase from Burkholderia cenocepacia J2315; ZP_04942934, putative triacylglycerol lipase from B. cenocepacia PC184; YP_625098, putative triacylglycerol lipase from B. cenocepacia AU 1054; YP_001778248, putative triacylglycerol lipase from B. cenocepacia MC0-3; YP_370846, putative triacylglycerol lipase from Burkholderia sp. strain 383. ZP_07149178, putative triacylglycerol lipase from Corynebacterium resistens DSM 45100; YP_119470, putative lipase from Nocardia farcinica IFM 10152. The amino acids constituting the conserved pentapeptide are underlined. Alignment was performed with the web tool MAFFT. Identical amino acids are on a black background, whereas a gray background indicates identical or equivalent amino acids.