Four residues of propeptide are essential for precursor folding of nattokinase

Abstract

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an a-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses.

Probing the Role of Catalytic Triad on the Cleavage between Intramolecular Chaperone and NK Mature Peptide

“…Type I IMCs include those that facilitate the formation of a tertiary structure, which is mostly produced in the form of N-terminal sequence extension; type II IMCs refer to those that do not directly participate in the formation of a tertiary structure but guides the assembly of a quaternary structure to form functional protein complexes, which are mostly located at the C-terminus of proteins. As shown in our previous study, − the folding of nattokinase (NK) is guided by its N-terminal propeptide that is a type I IMC.…”

Versatility of subtilisin: A review on structure, characteristics, and applications

“…29 The propeptide also provide structural information that contribute to building block of mature subtilisin functions during precursor folding in which mutations within the propeptide can alter the structure and activity of subtilisin, even though the mutation is not a part of the mature domain. 12 The quadruple Ala mutation, Y10A/G13A/G34A/G35A, made to residues of nattokinase propeptide has caused chaperone function loss for the propeptide as the residues have a part in forming the part of interface with subtilisin domain and are attached to an α-helix that contribute to the stability of propeptide structure. 12 In addition to that, mutation of leucine to proline residue in propeptide segment of a proform subtilisin homolog, pro-Tk-subtilisin, has also demonstrated a reduction in the binding of the propeptide to Tk-subtilisin, which in return accelerate the maturation of the subtilisinlike enzyme.…”

“…12 The quadruple Ala mutation, Y10A/G13A/G34A/G35A, made to residues of nattokinase propeptide has caused chaperone function loss for the propeptide as the residues have a part in forming the part of interface with subtilisin domain and are attached to an α-helix that contribute to the stability of propeptide structure. 12 In addition to that, mutation of leucine to proline residue in propeptide segment of a proform subtilisin homolog, pro-Tk-subtilisin, has also demonstrated a reduction in the binding of the propeptide to Tk-subtilisin, which in return accelerate the maturation of the subtilisinlike enzyme. 30 The maturation of subtilisin is also known to be dependent on the presence of calcium ions during the autolysis process as calcium has been stated by various studies to inhibit autolysis.…”

“…It is suggested that propeptide may be required as intramolecular chaperone to suppress denaturation of the later mature subtilisin molecules from mature subtilisin molecules that are activated earlier 29 . The propeptide also provide structural information that contribute to building block of mature subtilisin functions during precursor folding in which mutations within the propeptide can alter the structure and activity of subtilisin, even though the mutation is not a part of the mature domain 12 . The quadruple Ala mutation, Y10A/G13A/G34A/G35A, made to residues of nattokinase propeptide has caused chaperone function loss for the propeptide as the residues have a part in forming the part of interface with subtilisin domain and are attached to an α‐helix that contribute to the stability of propeptide structure 12 .…”

“…Propeptide domain is suggested by researchers as necessary for structural activity and the regulation of subtilisin. The domain is dubbed as the intramolecular chaperone, which according to Inouye in 1991 helps the folding of protein for formation of an active 3D structure, though it is not part of the mature segment of subtilisin and does not add to subtilisin function 12 . Scientists have also demonstrated that when aspartic residue (Asp 32) at the active site of subtilisin is substituted with asparagine, there was an absence of processing by the mutated prosubtilisin via in vitro experiments, which suggests that propeptide acts as an intramolecular regulator for activation of subtilisin 13 …”