A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex.

Lipases are hydrolytic enzymes which break down triacylglycerides into free fatty acids and glycerols. They have been classified as serine hydrolases owing to their inhibition by diethyl p-nitrophenyl phosphate. Lipase activity is greatly increased at the lipid-water interface, a phenomenon known as interfacial activation. X-ray analysis has revealed the atomic structures of two triacylglycerol lipases, unrelated in sequence: the human pancreatic lipase (hPL)4, and an enzyme isolated from the fungus Rhizomucor (formerly Mucor) miehei (RmL). In both enzymes the active centres contain structurally analogous Asp-His-Ser triads (characteristic of serine proteinases), which are buried completely beneath a short helical segment, or 'lid'. Here we present the crystal structure (at 3 A resolution) of a complex of R. miehei lipase with n-hexylphosphonate ethyl ester in which the enzyme's active site is exposed by the movement of the helical lid. This movement also increases the nonpolarity of the surface surrounding the catalytic site. We propose that the structure of the enzyme in this complex is equivalent to the activated state generated by the oil-water interface.

A serine protease triad forms the catalytic centre of a triacylglycerol lipase.

True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.

Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Rhizomucor miehei triglyceride lipase is synthesized as a precursor.

A Rhizomucor miehei cDNA library constructed in Escherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269 residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesized R. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing.

Partial purification and characterization of free and immobilized lipases fromMucor miehei.

An extracellular lipase, a glycoprotein, produced by fermentation with a selected strain ofMucor miehei has been partially purified in two forms, A and B. The two forms have a high degree of antigenic identity and have similar pH-activity profiles with tributyroylglycerol as a substrate with optima at pH 7. They differ as follows: A, in contrast to B, requires activation at alkaline pH before analysis; A binds with concanavalin-A more completely than B; the net charges are slightly different at pH 8; and the isoelectric points are different. Our results indicate that the B lipase is formed by partial deglycosylation of the A lipase and that this influences the activity toward emulsions.In addition, the two enzymes have been immobilized by adsorption. These preparations and the soluble forms were highly specific for primary esters of triacylglycerols (TG); they usually hydrolyzed TG of 12∶0, 14∶0, 16∶0, and 18∶1 more rapidly than those of 4∶0, 6∶0, and 8∶0 and 10∶0 in mixtures of monoacid TG (4∶0 to 18∶1); and they were not stereospecific for TG. Immobilization altered the specificity of the preparations somewhat, in that slightly more 14∶0 and 16∶0 were released.