Abstract
Many proteins are synthesized as precursors, with propeptides playing a variety of roles such as assisting in folding or preventing them from being active within the cell. While the precise role of the propeptide in fungal lipases is not completely understood, it was previously reported that mutations in the propeptide region of the Rhizomucor miehei lipase have an influence on the activity of the mature enzyme, stressing the importance of the amino acid composition of this region. We here report two structures of this enzyme in complex with its propeptide, which suggests that the latter plays a role in the correct maturation of the enzyme. Most importantly, we demonstrate that the propeptide shows inhibition of lipase activity in standard lipase assays and propose that an important role of the propeptide is to ensure that the enzyme is not active during its expression pathway in the original host.
Highlights
The lipase from the fungus Rhizomucor miehei (RmL refers to the mature enzyme, while ProRmL refers to the proenzyme) belongs to the family of triglyceride lipases (EC 3.1.1.3) called class 3 lipases (PF01764 in Pfam, https://pfam. xfam.org),[1] which are members of the α/β hydrolase superfamily (53474 in SCOP, http://scop.mrc-lmb.cam.ac. uk/scop/)[2] that hydrolyze the ester linkages of triglycerides
Sequence alignment of a selected set of fungal lipases with similarity to RmL reveals a similarity in the propeptide region, indicating a general phenomenon of inhibition of unwanted activities during the expression of the lipase in the host cell. These results clearly demonstrate the inhibitory effect of the isolated propeptide and indicate the structural basis for the inhibition and lower binding to the purification material
The mature domain is in the closed inactive form, and this is stabilized by a Article region of the propeptide that sits on top of the active site, preventing its opening
Summary
The lipase from the fungus Rhizomucor miehei (RmL refers to the mature enzyme, while ProRmL refers to the proenzyme) belongs to the family of triglyceride lipases (EC 3.1.1.3) called class 3 lipases (PF01764 in Pfam, https://pfam. xfam.org),[1] which are members of the α/β hydrolase superfamily (53474 in SCOP, http://scop.mrc-lmb.cam.ac. uk/scop/)[2] that hydrolyze the ester linkages of triglycerides. Class 3 lipases are found in animals, plants, protists, and prokaryotes and are only distantly related to other lipase families Their active sites were shown to contain a classical catalytic triad composed of Ser-His-Asp,[3] but in contrast to serine protease, the active sites are buried inside the structure. Class 3 lipases, similar to a number of other proteins, contain an “additional” region during folding, which is subsequently cleaved either by autoprocessing, as is the case for several proteases, or by external proteases. These regions, termed propeptides, are often referred to as intramolecular chaperones (see ref 5 and references therein) and, as such, are required for proper folding and activity of the mature protein. X-ray structures have been determined for a number of propeptides containing proteins, the proteases, as reviewed in ref 10
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