Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries.
This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.
Ichishima
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Department of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo, Japan. ichisima@t.soka.ac.jp
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology*; D001236:Aspergillus oryzae / Q000201:enzymology*; D001709:Biotechnology; D002268:Carboxypeptidases / Q000378:metabolism*; D002384:Catalysis; D043402:Cathepsin A; D002417:Cattle; D004765:Enteropeptidase / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D007218:Industrial Microbiology; D007564:Japan; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015032:Zinc / Q000378:metabolism
D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D006867:Hydrolases; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase; D004765:Enteropeptidase; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C029094:aspergillopepsin I; D008666:Metalloendopeptidases; D015032:Zinc; D008358:Mannose