Cloning, Expression, and Characterization of Tomato (Lycopersicon esculentum) Aminopeptidase P

Hauser, Felix; Strassner, Jochen; Schaller, Andreas

doi:10.1074/jbc.m103179200

Cited by 18 publications

(10 citation statements)

References 39 publications

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“…Although there are numerous papers monitoring changes in aminopeptidase activity during development (Walling and Gu, 1996), there are few studies examining aminopeptidase protein levels in plant development (Bartling and Nosek, 1994;Herbers et al, 1994;Hauser et al, 2001;Murphy et al, 2002). Therefore, it was of interest to more thoroughly examine the accumulation of the LAP-N, LAP-A and LAP-like proteins in vegetative and reproductive organs of tomato plants.…”

Section: Lap-n and Lap-a Protein Accumulation In Vegetative And Repromentioning

confidence: 99%

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Park

Walling

2003

Plant Physiology

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Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins. The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains. Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides. Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program. LapN was a single-copy gene encoding a rare-class transcript. A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A. Leucyl aminopeptidases (LAPs; EC 3.4.11.1) are members of the M17 family of peptidases (Barrett et al., 1998). LAPs are ubiquitous being found in animals, plants, and prokaryotic cells. These hexameric metallopeptidases catalyze the release of the N-terminal residues from protein, peptide, fluorometric, and chromogenic substrates. The best characterized LAPs are from Bos taurus, Escherichia coli and tomato (Lycopersicon esculentum). X-ray crystal structures of the bovine and E. coli LAPs have provided insight into the LAP catalytic mechanism (Kim and Lipscomb, 1994;Sträter and Lipscomb, 1995;Sträter et al., 1999a). The roles of selected residues of the E. coli and tomato LAPs in chromogenic or peptide substrate catalysis, respectively, have been tested by site-directed mutagenesis (Sträter et al., 1999b;Gu and Walling, 2002).In most plants, three classes of LAP-related polypeptides are detected using a tomato LAP antiserum, including the 66-and 77-kD LAP-like proteins and the 55-kD neutral LAP (LAP-N; Chao et al., 2000). Only in a subset of the Solanaceae is a second 55-kD LAP species (LAP-A) detected (Hildmann et al., 1992;Gu et al., 1996b;Chao et al., 2000). In tomato, LAP-A protomers have an acidic pI and are encoded by two genes (LapA1 and LapA2), which are expressed during floral and fruit development. The LapA genes are not expressed in foliage from healthy plants (Chao et al., 1999). However, LapA RNAs, proteins, and activities accumulate locally and systemically in leaves after wounding, Pseudomonas syringae pv. tomato and Phytophthora parasitica infection, and caterpillar feeding (Pautot et al., 1993(Pautot et al., , 2001Gu et al., 1996b;Chao et al., 1999;Jwa and Walling, 2001). The activation of LapA gene expression by jasmonic acid (JA), abscisic acid, the phytotoxin coronatine (a JA mimic), and suppression of LapA by salicylic acid is consistent with the regulation of the tomato LapA genes by the wound octadecanoid pathway (Chao et al., 1999). LapA genes also respond to signals generated during water deficit and salinity stress (Chao et al., 1999). The potato (Solanum tuberosum) Lap RNAs also ac...

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Section: Lap-n and Lap-a Protein Accumulation In Vegetative And Repromentioning

confidence: 99%

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Park

Walling

2003

Plant Physiology

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“…Since the time an enzyme with the specificity of APP was first purified from Escherichia coli (24), APPs have been characterized from diverse sources, including bacteria (16), nematodes (15), insects (14), plants (10), and tissues from several mammalian species (9,11). While the physiological role of APP in bacteria is unclear, mammalian APP is involved in the protein turnover of collagen and the regulation of biologically active peptides, such as substance P and bradykinin (5,23,26).…”

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confidence: 99%

Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1

Lee

Kim

Bae

et al. 2006

Appl Environ Microbiol

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Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(N -Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (K m , 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co 2؉ -, Mn 2؉ -, or Zn 2؉ -dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 min at 80°C. This study represents the first characterization of a hyperthermophilic archaeon APP.Aminopeptidase P (APP, or X-Pro aminopeptidase; EC 3.4.11.9) is a peptidase that specifically removes the N-terminal amino acids from peptides in which the penultimate residue is proline (5). Since the time an enzyme with the specificity of APP was first purified from Escherichia coli (24), APPs have been characterized from diverse sources, including bacteria (16), nematodes (15), insects (14), plants (10), and tissues from several mammalian species (9, 11). While the physiological role of APP in bacteria is unclear, mammalian APP is involved in the protein turnover of collagen and the regulation of biologically active peptides, such as substance P and bradykinin (5,23,26). It has been shown that APPs from a number of lactococcal strains may contribute to the abolition of bitterness during the ripening of cheese by participating in peptide degradation following release into the cheese matrix (17).To date, however, there have been no reports on the properties of an APP from either an archaeon or a hyperthermophile. With the availability of a generally applicable combination of conventional genetic engineering and genomic research techniques, the genome sequences of some hyperthermophilic microorganisms are of considerable biotechnological interest because of their heat-stable enzymes, and many extremely thermostable enzymes are being developed for biotechnological purposes (22). Furthermore, recent advances in the application of molecular biological tools to hyperthermophilic archaea, such as gene knockout techniques and efficient transformation systems, could facilitate the study of hyperthermophilic archaeal gene function and contribute to an understanding of the physiology of hyperthermophilic archaea.To facilitate the search for valuable and extremely thermostable enzymes and to help answer questi...

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“…The constructs were transformed into E. coli BL21 codon plus (DE3)-RIL (Stratagene). The fusion proteins were purified from IPTG-induced cultures by affinity chromatography on immobilized glutathione and analyzed by SDS-PAGE as described (Hauser et al, 2001). …”

Section: Lecpk1-l and -Smentioning

confidence: 99%

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization

et al. 2002

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The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2,K m = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of theLeCPK1 N terminus was found to be required for plasma membrane targeting.

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Cloning, Expression, and Characterization of Tomato (Lycopersicon esculentum) Aminopeptidase P

Cited by 18 publications

References 39 publications

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization

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