title: Large propeptides of fungal beta-N-acetylhexosaminidases are novel enzyme regulators that must be intracellularly processed to control activity, dimerization, and secretion into the extracellular environment.
abstract: Filamentous fungi produce and secrete beta-N-acetylhexosaminidases, Hex, as important components of the binary chitinolytic systems involved in the formation of septa and hyphenation. Enzyme reconstitution experiments published previously indicate that Hex can occur in the form of two molecular species containing either one or two molecules of the propeptide noncovalently associated with the enzyme dimer. Here, we describe a novel mechanism for the regulation of the activity of Hex based on the association of their catalytic subunits with the large N-terminal propeptides in vivo. We show that the enzyme precursor is processed early in the biosynthesis, shortly after the addition of N-glycans through the action of a dibasic peptidase, cleaving both before and after the dibasic sequence. The processing site for this unique dibasic peptidase, different from that of kexins, is conserved among the beta-N-acetylhexosaminidases from filamentous fungi, and inhibition of the dibasic peptidase abrogates enzyme folding and activation. Binding of the released propeptide to the catalytic subunit of Hex is essential for its activation. An examination of the kinetics of Hex activation and dimerization in vitro allowed us to understand the unusually high efficiency of the assembly of this enzyme. We also report that the fungus is able to actively regulate the concentration of the processed propeptide in endoplasmic reticulum and thus the specific activity of the produced Hex. This novel regulatory mechanism enables the control of the catalytic activity and architecture of the secreted enzyme according to the needs of the producing cell at various stages of its growth cycle.
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, VídenskA 1083, 14220 Praha 4, Czech Republic.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Plíhal
authors.forename: Jan
authors.initials: J
authors.lastname: Sklenár
authors.forename: Katerina
authors.initials: K
authors.lastname: Hofbauerová
authors.forename: Petr
authors.initials: P
authors.lastname: Novák
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Daniel
authors.initials: D
authors.lastname: Kavan
authors.forename: Helena
authors.initials: H
authors.lastname: Ryslavá
authors.forename: Lenka
authors.initials: L
authors.lastname: Weignerová
authors.forename: Andrea
authors.initials: A
authors.lastname: Charvátová-Pisvejcová
authors.forename: Vladimír
authors.initials: V
authors.lastname: Kren
authors.forename: Karel
authors.initials: K
authors.lastname: Bezouska
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D001692:Biological Transport; D002384:Catalysis; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004795:Enzyme Stability; D005658:Fungi / Q000201:enzymology*; D045683:Furin / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013844:Thiazoles / Q000494:pharmacology; D014158:Transcription, Genetic; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D013844:Thiazoles; D001619:beta-N-Acetylhexosaminidases; D045683:Furin; C487818:N-acetylglucosamine thiazoline; D000117:Acetylglucosamine