title: Heterologous expression and site-directed mutagenesis studies on the activation mechanism and the roles of the basic residues in the prosegment of aspergillopepsinogen I.
abstract: To study the structure/function relationship of the prosegment of aspartic proteinase, a putative proform of aspergillopepsin I (or proteinase B) from Aspergillus niger var. macrosporus was expressed by Escherichia coli, refolded in vitro, and purified. The conversion of the purified proenzyme (aspergillopepsinogen I, proproteinase B) into the active mature form occurred at pH < or = 4.5 and was completely inhibited by pepstatin A, a specific inhibitor for aspartic proteinase, suggesting autoprocessing. The N-terminus of this mature form was Glu67 (numbering in preproform), which was different from the N-terminal Ser70 of native proteinase B although there was no significant difference in enzymatic activity. During the conversion, two intermediates were observed on SDS/PAGE, indicating a stepwise mechanism. The Lys56-Phe57 sequence seems to be a counterpart of the Lys-Tyr pair highly conserved in the prosequences of aspartic proteinases. When the mutant proenzyme (K56N), in which Lys56 was replaced with Asn by site-directed mutagenesis, was allowed to refold under various conditions, no significant potential activity could be obtained. Proproteinase B was also expressed by Bacillus brevis HPD31. This system required no in vitro refolding to obtain potentially active proenzyme, which was secreted into the culture medium (30-120 mg/l) and had the same properties with that obtained by the E. coli system. The K56N mutant prepared by this system also had no potential activity, and was rapidly digested by incubation with native proteinase B, suggesting that the mutant did not fold correctly. On the other hand, the K56R mutant (Lys56-Arg) was potentially active. These results indicated that Lys56 is essential for the folding through electrostatic interaction with the catalytic Asp residues in the active site although it may be replaced with Arg. In the presence of a low concentration of pepstatin A, an incompletely processed form with N-terminal Ser53 was obtained. Further, the R52Q (Arg52-->Glin) mutant showed no processing but was converted to the active mature form by incubation with the native enzyme. Therefore, the cleavage between Arg52 and Ser53 is considered to be the initial and essential step of the autoactivation. The R26Q, K27Q, R36Q, K40Q, R42Q, and K66Q mutants were also potentially active. The K66Q mutant was processed to a form with N-terminal Ala55.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: T
authors.initials: T
authors.lastname: Hayashi
authors.forename: X P
authors.initials: XP
authors.lastname: Huang
authors.forename: J F
authors.initials: JF
authors.lastname: Lu
authors.forename: S B
authors.initials: SB
authors.lastname: Athauda
authors.forename: K H
authors.initials: KH
authors.lastname: Kong
authors.forename: H
authors.initials: H
authors.lastname: Yamagata
authors.forename: S
authors.initials: S
authors.lastname: Udaka
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D001407:Bacillus / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D004271:DNA, Fungal; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins