title: 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 α-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.
authors.affiliation: Beijing Key Laboratory of Plants Resource Research and Development, School of Science, Beijing Technology and Business University, Beijing 100048, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jia
authors.affiliation: State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Bao
authors.affiliation: State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Changyan
authors.initials: C
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Hanjing
authors.initials: H
authors.lastname: Ding
authors.affiliation: State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China zouguolin@whu.edu.cn.
authors.forename: Zhongliang
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China zouguolin@whu.edu.cn.
authors.forename: Guolin
authors.initials: G
authors.lastname: Zou
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D013381:Subtilisins; C053771:nattokinase
keywords: essential residues; intramolecular chaperone; nattokinase; propeptide; subtilisin