Ib Svendsen

Barley α-amylase/subtilisin inhibitor. I. Isolation and characterization

A protein inhibitor of endogenous α-amylase 2 has been isolated from germinated barley by glycogen precipitation followed by cation-exchange chromatography. Preliminary kinetic analysis showed a mixed type mechanism of inhibition with an apparent Ki of 4×10−8M. The inhibitor formed well-defined complexes with barley malt α-amylase 2 and co-purified with the α-amylase by cycloheptaamylose affinity chromatography of glycogen precipitates. The inhibitor was inactive towards α-amylases from sorghum malt, hog pancreas, Aspergillus oryzae, and Bacillus subtilis. The amino acid composition and molecular weight near 21,000 were found to be the same as those of both “band-2 protein” previously identified in preparations of barley malt α-amylase and a specific subtilisin inhibitor from barley. Inhibition experiments confirmed that the malt α-amylase inhibitor is a strong inhibitor of subtilisin Carlsberg. Measurements of α-amylase activity in the presence of equimolar amounts of inhibitor and subtilisin showed that the inhibitor is “double headed”. The inhibitory activity towards α-amylase was lost after treatment of the inhibitor at 70 °C for 15 min. Isoelectric focusing patterns confirmed that the partially heat-labile, basic α-amylase isozymes (pI=6.6) of barley malt are complexes of α-amylase 2 (pI=6.2) and the inhibitor (pI=7.2). Evidence is presented to suggest that proteins with properties similar to those of the barley inhibitor are present in other cereals including wheat and rye. Possible in vivo functions and some practical aspects of the barley inhibitor are discussed.

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin: cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes.

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of l-valine andl-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast,Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes l-valine as well asl-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k c value with l-valine as substrate than with l-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. BothCYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.

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

The primary structure of glucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of G1 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 G1. Sequencing of glucoamylase G1 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 G1 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 α-amylases.

Two antifungal thaumatin-like proteins from barley grain

Antifungal activity has been associated with 2 immunochemically distinct proteins, protein R and S (M r ∼23 kDa; pI 9‐10), which were isolated in pure form from barley grain. The proteins are homologous with thaumatin‐ and pathogenesis‐related proteins of the PR5 family. The proteins inhibit growth of i.a. Trichoderma viride and Candida albicans in microtiter plate assays and act synergistically with barley grain chitinase C. Like maize zeamatin, protein R and S but not chitinase C retarded fungal growth in synergism with nikkomycin Z, a nucleoside‐peptide inhibitor of fungal chitin synthesis. Although no inhibition of α‐amylases or serine proteases could be associated with protein R or S the results indicate that the homologous maize grain bifunctional inhibitor of insect α‐amylase and trypsin is very similar to or identical with maize zeamatin, which was proposed to have permeabilizing activity towards fungal membranes. Thus, in addition to the intensely sweet properties of thaumatin, multiple unrelated defense functions against insect and fungal pests can now be associated with the family of thaumatin‐homologous proteins.

The primary structure of noxiustoxin: A K+ channel blocking peptide, purified from the venom of the scorpion Centruroides noxius Hoffmann

Noxiustoxin, component II-11 from the venom of scorpion Centruroides noxiusHoffmann, was obtained in pure form after fractionation by Sephadex G-50 chromatography followed by ion exchange separation on carboxy-methylcellulose columns (17). The primary structure of Noxiustoxin, a polypeptide 39 amino acid residues long was determined by automaticEdman degradation and chemical cleavage with cyanogen bromide followed by amino acid analysis of the two resulting peptides. Its sequence is: Thr−Ile−Ile−Asn−Val−Lys−Cys−Thr−Ser−Pro−Lys−Gln−Cys−Ser−Lys−Pro−Cys−Lys−Glu−Leu−Tyr−Gly−Ser−Ser−Ala−Gly−Ala−Lys−Cys−Met−Asn−Gly−Lys−Cys−Lys−Cys−Tyr−Asx−Asn, with a molecular weight of 4,184±6. No histidine, arginine, tryptophan or phenylalanine was found. Noxiustoxin is the first short toxin directed against mammals and the first K+ channel blocking polypeptide-toxin (4) found in scorpion venoms.

Characterization of two forms of glucoamylase from aspergillus niger

Aspergillus niger glucoamylases GI and GII (E.C. 3.2.1.3) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography. Both enzymes consist of a single glycosylated polypeptide chain. The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000. The amino acid compositions of GI and GII were very similar. Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms. In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxillary protease were analyzed by high pressure gel permeation chromatography. The elution profiles were also consistent with GI and GII having similar polypeptide chains. However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms.

Isolation and Reconstitution of Cytochrome P450ox and in Vitro Reconstitution of the Entire Biosynthetic Pathway of the Cyanogenic Glucoside Dhurrin from Sorghum

A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside [beta]-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-[alpha]-dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled.

Characterization of a bifunctional wheat inhibitor of endogenous α-amylase and subtilisin

A bifunctional α‐amylase/serine protease inhibitor which inhibits germination‐specific cereal α‐amylases of the Graminae subfamily Festucoideae as well as bacterial subtilisins has been isolated from wheat grains. This protein has M r ≈20500 and pI ≈7.2. The amino acid composition and N‐teminal sequence (45 residues) show that the inhibitor is homologous with cereal and leguminous inhibitors of the soybean trypsin inhibitor (Kunitz) family.

The complete amino acid sequence of copper, zinc superoxide dismutase from Saccharomyces cerevisiae

The amino acid sequence of the copper zinc superoxide dismutase from Saccharomyces cerevisiae has been determined by automated Edman degradation. Peptides were obtained from cyanogen bromide cleavage, Staphylococcus aureus V8 protease digestion, tryptic and chymotryptic digests of the citraconylated reduced and carboxymethylated enzyme, and by further fragmentation of selected peptides with trypsin. From the alignment of these peptides and the previously published sequence of the first 54 amino terminal residues (24) the complete sequence was deduced by direct sequence identification of all 153 amino acid residues and of all peptide overlaps. The amino acid sequence corresponds to a molecular weight of 15,950 for each of the two identical subunits in the native enzyme. The primary structure of yeast copper, zinc superoxide dismutase is 55% identical with the sequence of the copper, zinc enzyme from bovine erythrocytes. Importantly, all the copper and zinc ligands, six histidine residues and one aspartate residue from the bovine enzyme, are conserved in the yeast enzyme. The high overall sequence homology and conservation of important metal binding active site amino acid residues suggest that the three-dimensional structure and in particular the active site geometry is virtually the same for the bovine and yeast enzyme. In contrast no sequence homology is apparent by comparison with the manganese or iron class of superoxide dismutases indicating that the two classes have not evolved from a common ancestor.

Sequence homology between barley endosperm protein Z and protease inhibitors of the α1-antitrypsin family

Six cDNA clones encoding parts of protein Z, a major barley endosperm albumin, have been identified. Nucleotide and amino acid sequencing have established a 180 residues long C‐terminal amino acid sequence of protein Z as well as two minor amino acid sequences (14 and 7 residues). These sequences show that barley protein Z is homologous with human α1‐antitrypsin, human otj‐antichymotrypsin, human antithrombin III, mouse contrapsin and chicken ovalbumin (26–32% of the 180 residues in the C‐terminal sequence in identical positions). The sequence homology and specific cleavage of protein Z at a bond corresponding to the reactive site of the inhibitors indicate a possible inhibitory function. Inhibition of microbial or pancreatic serine proteases could, however, not be associated with protein Z.