The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Abstract

The primary structure ofglucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of GI were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517-527 (1983)), identification of 574 of the 614 amino acid residues in Gl. Sequencing of glucoamylase Gl cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.The majority of the carbohydrate of Gl is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various at-amylases.Abbreviations: BNPS-skatole = 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; ca = citraconyl; Con A = concanavalin A; DFP = diisopropylfluorophosphate; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediaminetetraacetic acid, disodium salt; Hepes = N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; Nemac = N-ethylmorpholine acetate; HPLC = high pressure liquid chromatography; PTH = phenylthiohydantoin; SDS-PAGE = polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; 2-pe = 2-pyridylethyl; G I designates the larger and G2 the smaller of the forms ofglucoamylase from A. niger (44).

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

“…The UV-absorption of the isolated peptide (Figure 3) confirmed the presence of an intact tryptophan in the fragment from G2 oxidized in the presence of acarbose, while in contrast the fragment from the inactive G2 derivative exhibited oxindolealanine UV-absorption. Minor components from digests of both inactive and active G2 derivatives comprising residues Phe(109)-Arg(122) and Phe(109)-Arg(125) (30) were also isolated and possessed similar UVspectra to those above (results not shown). Thus, the oxidation of the Trp(120) seemed to be accompanied by the loss of maltose-hydrolyzing capacity of glucoamylase.…”

“…10% of non-ligand binding enzyme molecules which were eliminated by affinity chromatography on acarbose-Sepharose (section 3.2). The tryplic fragments of G2 deriv- in the amino acid sequence ofglucoamylase (5,30). The UV-absorption of the isolated peptide (Figure 3) confirmed the presence of an intact tryptophan in the fragment from G2 oxidized in the presence of acarbose, while in contrast the fragment from the inactive G2 derivative exhibited oxindolealanine UV-absorption.…”

“…100 amino acid residues, bul the two forms have essentially the same activity on soluble substrates (31). The amino acid sequence of the enzyme (5,6,30) near the Abbreviations: AH = aminohexyl; G1 and G2 = the larger and the smaller of the two forms ofglucoamylase from A. niger (31); HPLC = high pressure liquid chromatography; NBS = N-bromosuecinimide; TFA = trifluoroacetic acid; Tris = 2-amin~-2(hydroxymethyl)-l, 3-propandioI. essential residue resembles that near a tryptophanyl residue of Taka-amylase A, proposed to interact with substrate in the active site cleft of this a-amylase (20,33). O.Oz Fine Chemicals, Uppsala, Sweden.…”

“…Bio-Gel P-100 was from Bio-Rad, Richmond, CA. Substrates and inhibitors were obtained as earlier reported (9) as were reagents and solvents for ~0.0( preparation, isolation and sequencing ofpeptide ~: fragments (29,30). x~ 3.…”

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

“…(30) and A. saitoi (27) in the presence of substrates or inhibitors has previously led to the suggestion that the essential carboxyl groups are located at or near subsites 2 and 3. Crystals suitable for structural analysis have not been obtained from a glucoamylase, but substantial sequence homology exists (29,53) between the enzymes from A. niger (6,50,51), Rh. oryzae (4), S. cerevisiae (diastaticus) (60), Saccharomycopsis fibuligera (29) and S. cerevisiae (59).…”