cDNA, amino acid and carbohydrate sequence of barley seed-specific peroxidase BP 1

Abstract

The major peroxidase of barley seed BP 1 was characterized. Previous studies showed a low carbohydrate content, low specific activity and tissue-specific expression, and suggested that this basic peroxidase could be particularly useful in the elucidation of the structure-function relationship and in the study of the biological roles of plant peroxidases (S.K. Rasmussen, K.G. Welinder and J. Hejgaard (1991) Plant Mol Biol 16: 317-327). A cDNA library was prepared from mRNA isolated from seeds 15 days after flowering. Full-length clones were obtained and showed 3' end length variants, a G+C content of 69% in the translated region, a 90% G or C preference in the wobble position of the codons and a typical signal peptide sequence. N-terminal amino acid sequencing and sequence analysis of tryptic peptides verified 98% of the sequence of the mature BP 1 which contains 309 amino acid residues. BP 1 is the first characterized plant peroxidase which is not blocked by pyroglutamate. BP 1 polymorphism was observed. BP 1 is less than 50% identical to other plant peroxidases which, taken together with its developmentally dependent expression in the endosperm 15-20 days after flowering, suggests a unique biological role of this enzyme. The barley peroxidase is processed at the C-terminus and might be targeted to the vacuole. The single site of glycosylation is located near the C-terminus in the N-glycosylation sequon -Asn-Cys-Ser- in which Cys forms part of a disulphide bridge. The major glycan is a typical plant modified-type structure, Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc. The BP 1 gene was RFLP-mapped on barley chromosome 3, and we propose Prx5 as the name for this new peroxidase locus.

Effect of Calcium, Other Ions, and pH on the Reactions of Barley Peroxidase with Hydrogen Peroxide and Fluoride

“…The protein product is very stable, however, and BP 1 is the most abundant peroxidase of mature barley grain. Protein signals target BP 1 for the secretory pathway (N-terminal signal peptide) and onward to the vacuole (Cterminal propeptide) (37). In the present work, we document that plant peroxidase activity can also be strongly regulated at the enzyme level through protein conformational changes induced by pH and Ca 2ϩ .…”

Isolation and Molecular Cloning of a Major Wheat Allergen, Tri a Bd 27K

“…The major allergens responsible for bakers' asthma, contained in the salt-soluble fraction of wheat, have been found to be -amylase inhibitors. 3,4) Weiss et al, 5) Posch et al 6) and Sander et al 7) found that other allergens connected with bakers' asthma occur in the water/salt-soluble fraction (albumins and globulins) of wheat proteins. Nine allergens with molecular masses of [14][15][16][17][18][26][27][28], and 35 kDa, were identified asamylase/protease inhibitors, acyl-CoA oxidase and fructose-bisphosphate aldolaseres from wheat, barley, maize, and rice by two-dimensional gel electrophoresis, immunoblotting, and N-terminal amino acid sequencing.…”

In vitro evolution of horse heart myoglobin to increase peroxidase activity

“…For this reason, the expression of functional, recombinant peroxidases and the design of novel peroxidases with altered substrate specificity are attractive goals. In the past decade, an increasing number of peroxidases from various sources has been cloned (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) and studied by sitedirected mutagenesis (17)(18)(19)(20). However, peroxidases invariably are expressed in Escherichia coli as either soluble apoproteins or as heme-deficient inclusion bodies (21,22).…”