Molecular and Structural Characterizations of Lipases from Chlorella by Functional Genomics

Abstract

Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/β hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

Keywords: Chlorella; enzymes; lipases; molecular modeling.

Figure 1

3D models of seven putative lipases without transmembrane domains. Four of them (Lip_1704, Lip_1795, Lip_4364, Lip_6297) display only the core module with a Rossman Fold architecture. Lip_3448 presents a C2 N-terminal domain, while Lip_5800 and Lip_5999 present a C-terminal alpha helices module. Lids are shown in dark blue and active site serine in yellow sticks.

Figure 2

(a) Three predicted membrane-associated lipases with a transmembrane module shown in lilac; lids are shown in dark blue and active site serine in yellow sticks. (b) Gene annotation and domain boundaries of Lip_4551 (left panel), Qmeanbrane result for transmembrane localization for Lip_4551 (right panel).

Figure 3

Multiple sequence alignment of putative lipases showing the conserved lipase 3 motif GXSXG and the conserved G residue for GX classification highlighted with orange star.

Figure 4

(a) Slabbed close up view of the active site cavity for Lip_1704 showing a crevice-like shape (b) A surface top view with DEPTH showing the shape of substrate entrance in the same protein.

Figure 5

(a) 3D model of putative Lysosomal acid lipase Lip_5462 lid is shown in dark blue and catalytic serine in yellow sticks. (b) Crystal structure of human Lysosomal acid lipase PDB ID: 6V7N. Lid is shown in orange and catalytic serine in yellow sticks. (c) The overlay of the two aforementioned structures showing high structure similarities and lid differences. (d) A close up view of the three helices lid of Lip_5462 showing conserved cysteine residues in red sticks.