Identification of a plant aminopeptidase with preference for aromatic amino acid residues as a novel member of the prolyl oligopeptidase family of serine proteases

Abstract

Genome analysis has indicated that plants, like animals, possess a variety of protease genes. However, bulk of putative proteases has not been characterized at the enzyme level. In this article, a novel enzyme that hydrolyses phenylalanyl-4-methylcoumaryl 7-amide (phenylalanyl-MCA) was purified from cotyledons of daikon radish by ammonium sulphate fractionation and successive chromatography with DEAE-cellulose, phenyl-Sepharose, Sephacryl S-200 and Mini-Q. The molecular mass of the enzyme was estimated to be 78 kDa by SDS-PAGE under reducing conditions and 74 kDa by gel filtration, indicating that the enzyme is a monomer. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that the enzyme is an orthologue of Arabidopsis unidentified protein, acylpeptide hydrolase-like protein (AHLP; UniProt ID: Q9FG66) belonging to the prolyl oligopeptidase (POP) family of a serine-type peptidase predicted from genetic information. Good substrates identified for the enzyme include phenylalanyl-MCA, tyrosyl-MCA and enkephalin. Neither acylamino acid-releasing activity nor endopeptidase activity was detected. The enzyme cleaved enkephalin (YGGFM, YGGFL), whereas, BAM-12 P (YGGFMRRVGRPE) and dynorphin A (YGGFLRRIRPKLK) were not digested. These results suggested that the enzyme possesses strict size selectivity of substrate. We propose the name 'tyrosyl aminopeptidase' for the uncharacterized protein AHLP.

Oilseed rape (Brassica napus): the importance of aminopeptidases in germination under normal and heavy metals stress conditions

“…22 Several proteases, especially aminopeptidases from other plants, were reported to show similar temperature optima. 12,[35][36][37][38] The pH profile of aminopeptidases activity and the pH optimum (Supporting Information, Fig. S6) for Phe-pNA was similar to that obtained for dry seeds of oilseed rape (pH 6.5 and smaller peak at pH 9.0).…”

The Plastid and Mitochondrial Peptidase Network in Arabidopsis thaliana: A Foundation for Testing Genetic Interactions and Functions in Organellar Proteostasis

“…In the same study, it was suggested that FTSH3/10 also accumulate as heteromers. However, they do not coexpress in our network; in fact, FTSH10 has one shared coexpressor with inner mitochondrial membrane OMA1 (module IX) ( Figure 5A), whereas FTSH3 shares two coexpressors with an uncharacterized M16 peptidase AT5G56730 (the same family as SPP, MPP1/2, and PREP1/2) and one with uncharacterized S9 peptidase AARE2/AHLP, the ortholog of S9 tyrosine amino peptidase in daikon radish; Tsuji et al, 2011) (Figure 4). Mitochondria possess a third FTSH member, FTSH4, with the active site facing the intramembrane space.…”

Recruitment of an ancient branching program to suppress carpel development in maize flowers

“…ANAC087, with ANAC046, initiates programmed cell death in the root cap, and regulates chromatin degradation following programmed cell death (34). Two proteases show a similar pattern of regulation: a cysteine protease (GRMZM2G456217) with an arabidopsis homolog (CEP1) with roles in programmed cell death of xylem elements and the tapetum (35, 36), and an uncharacterized serine protease (GRMZM2G313321, 37). These and other examples (Table 1) are consistent with roles for GT1 and RA3 in carpel suppression, and further point to genes that may be directly or indirectly regulated by GT1 and RA3.…”