Leucyl aminopeptidase
Encyclopedia
Leucyl aminopeptidases are enzymes that preferentially catalyze the hydrolysis
of leucine
residues
at the N-terminus of peptides and proteins. Other N-terminal residues can also be cleaved, however. LAPs have been found across superkingdoms
. Identified LAPs include human LAP, bovine lens LAP, porcine LAP, Escherichia coli (E. coli) LAP (also known as PepA or XerB), and the solanaceous-specific acidic LAP (LAP-A) in tomato
(Solanum lycopersicum).
s in PepA and in bovine lens LAP have been found to be similar. Shown in the picture below is the proposed model for the active site of LAP-A in tomato based on the work of Strater et al. It is also known that the biochemistry
of the LAPs from these three kingdoms
is very similar. PepA, bovine lens LAP, and LAP-A preferentially cleave N-terminal leucine, arginine
, and methionine
residues. These enzymes are all metallopeptidases requiring divalent
metal
cations
for their enzymatic activity Enzymes are active in the presence of Mn+2, Mg+2 and Zn+2. These enzymes are also known to have high pH
(pH 8) and temperature
optima. At pH 8, the highest enzymatic activity is seen at 60oC. PepA, bovine lens LAP and LAP-A are also known to form hexamer
s in vivo
. The Gu et al. from 1999 demonstrated that six 55kDA enzymatically inactive LAP-A protomer
s come together to form the 353kDa bioactive LAP-A hexamer. Structures of the bovine lens LAP protomer and the biologically active hexamer have been constructed can be found through Protein Data Bank (2J9A).
of carboxypeptidase
s and endoprotease
have been much more well-studied and understood by researchers (Ref #6 Lipscomb 1990). Work within the past two decades has provided vital knowledge regarding the mechanisms of aminopeptidases. The mechanism of
bovine lens LAP and PepA have been elucidated (Ref 1 and 2), however, the exact mechanism of tomato LAP-A is unknown at this time. A search of current literature does not indicate that new research is underway to determine the exact mechanism of LAP-A. Based on the biochemical similarities of the LAPs between kingdoms, the mechanism of LAP-A may be similar to bovine lens LAP and PepA.
Once thought of as a housekeeping gene
necessary only for protein turnover
, studies have demonstrated that LAP-A has a regulatory role in the immune response in tomato.
In order to survive, plants must be able to respond to many biotic
and abiotic stresses, including pathogen
attack, piercing/sucking
insect
s, herbivory
, and mechanical wounding. These stresses activate specialized signal transduction pathways
, which are specific to the stressor and the amount of tissue damage inflicted. Similar to mechanical wounding, chewing insects, such as the tobacco hornworm (Manduca sexta, one of the major pests of tomato), cause extensive tissue
damage activating the jasmonic acid
(JA)-mediated response (Walling 2000). This JA-mediated response revolves around the octadecadnoid pathway, which is responsible for the synthesis of JA and several other potent signaling molecules, and ends in the regulation of two temporal
ly distinct sets of genes
. The early genes amplify the wounding signal and can be detected 30 minutes to 2 hours after damage (Ryan 2000). Late gene expression can be seen 4–24 hours after wounding. Products of late-response genes act as deterrents to chewing-insect feeding, often by decreasing the nutritional value of the food ingested or interfering with insect gut function (Walling 2000). For example, serine
proteinase inhibitors (Pins) interfere with digestive proteases in the insect gut and polyphenol oxidase
s (PPO) act to decrease the nutritive value of plant leaves after ingestion
by herbivores (Johnson et al. 1989; Ryan 2000; Orozco-Cardenas 2001). Please see the Picture 3 for a summary of the wound response in tomato.
The plant response in this octadecanoid pathway is similar to mammalian prostaglandin
and
leukotriene
pathways (Ref Walling 2000). This particular pathway is inhibited by salicylic acid
.
(LAP-A), a product of the octadecanoid pathway in some solanaecous plants, has been shown by Fowler et al. to have a regulatory role in the late wound response of tomato. Experiments were conducted using three genotypes of tomato plants: wildtype (WT), (LapA-SI) plants that were silenced for LAP-A, and LapA-OX that constitutively expressed LAP-A. Late-gene expression was inhibited in wounded LapA-SI plants, and the LapA-SI plants were also more susceptible to tobacco hornworm feeding, relative to wildtype (WT) plants. In comparison, the wounded LapA-OX leaves exhibited heightened levels of late gene RNA accumulation, an increased resistance to herbivory, and extended expression of late wound-response genes. These data suggest that LAP-A functions in regulating both the intensity and the persistence of the late wound response. However, unwounded LapA-OX did not accumulate late gene RNA
transcripts, suggesting that presence of LAP-A alone is not sufficient to induce late gene expression. LAP-A is the first plant aminopeptidase shown to have a regulatory role in signal transduction pathway.
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of leucine
Leucine
Leucine is a branched-chain α-amino acid with the chemical formula HO2CCHCH2CH2. Leucine is classified as a hydrophobic amino acid due to its aliphatic isobutyl side chain. It is encoded by six codons and is a major component of the subunits in ferritin, astacin and other 'buffer' proteins...
residues
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
at the N-terminus of peptides and proteins. Other N-terminal residues can also be cleaved, however. LAPs have been found across superkingdoms
Domain (biology)
In biological taxonomy, a domain is the highest taxonomic rank of organisms, higher than a kingdom. According to the three-domain system of Carl Woese, introduced in 1990, the Tree of Life consists of three domains: Archaea, Bacteria and Eukarya...
. Identified LAPs include human LAP, bovine lens LAP, porcine LAP, Escherichia coli (E. coli) LAP (also known as PepA or XerB), and the solanaceous-specific acidic LAP (LAP-A) in tomato
Tomato
The word "tomato" may refer to the plant or the edible, typically red, fruit which it bears. Originating in South America, the tomato was spread around the world following the Spanish colonization of the Americas, and its many varieties are now widely grown, often in greenhouses in cooler...
(Solanum lycopersicum).
Enzyme Description, Structure, and Active Site
The active siteActive site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
s in PepA and in bovine lens LAP have been found to be similar. Shown in the picture below is the proposed model for the active site of LAP-A in tomato based on the work of Strater et al. It is also known that the biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
of the LAPs from these three kingdoms
Kingdom (biology)
In biology, kingdom is a taxonomic rank, which is either the highest rank or in the more recent three-domain system, the rank below domain. Kingdoms are divided into smaller groups called phyla or divisions in botany...
is very similar. PepA, bovine lens LAP, and LAP-A preferentially cleave N-terminal leucine, arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
, and methionine
Methionine
Methionine is an α-amino acid with the chemical formula HO2CCHCH2CH2SCH3. This essential amino acid is classified as nonpolar. This amino-acid is coded by the codon AUG, also known as the initiation codon, since it indicates mRNA's coding region where translation into protein...
residues. These enzymes are all metallopeptidases requiring divalent
Divalent
In chemistry, a divalent ion or molecule has a valence of two and thus can form two bonds with other ions or molecules. An older term for divalent is bivalent....
metal
Metal
A metal , is an element, compound, or alloy that is a good conductor of both electricity and heat. Metals are usually malleable and shiny, that is they reflect most of incident light...
cations
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
for their enzymatic activity Enzymes are active in the presence of Mn+2, Mg+2 and Zn+2. These enzymes are also known to have high pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
(pH 8) and temperature
Temperature
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...
optima. At pH 8, the highest enzymatic activity is seen at 60oC. PepA, bovine lens LAP and LAP-A are also known to form hexamer
Hexamer
A hexamer is a thing composed out of six sub-units.In microbiology, a hexamer is one of the proteins composing the polyhedral protein shell that encloses the bacterial micro-compartments known as carboxysomes....
s in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
. The Gu et al. from 1999 demonstrated that six 55kDA enzymatically inactive LAP-A protomer
Protomer
In structural biology, a protomer is the structural unit of an oligomeric protein. A protomer can be a protein subunit or several different subunits, that assemble in a defined stoichiometry to form an oligomer. The protomer is the smallest subset of different subunits that form the oligomer. The...
s come together to form the 353kDa bioactive LAP-A hexamer. Structures of the bovine lens LAP protomer and the biologically active hexamer have been constructed can be found through Protein Data Bank (2J9A).
LAP Mechanism(s)
Historically, the mechanismsReaction mechanism
In chemistry, a reaction mechanism is the step by step sequence of elementary reactions by which overall chemical change occurs.Although only the net chemical change is directly observable for most chemical reactions, experiments can often be designed that suggest the possible sequence of steps in...
of carboxypeptidase
Carboxypeptidase
A carboxypeptidase is a protease enzyme that hydrolyzes the peptide bond of an amino acid residue at the carboxy-terminal end...
s and endoprotease
Endopeptidase
Endopeptidase or endoproteinase are proteolytic peptidases that break peptide bonds of nonterminal amino acids , in contrast to exopeptidases, which break peptide bonds from their end-pieces. For this reason, endopeptidases cannot break down peptides into monomers, while exopeptidases can break...
have been much more well-studied and understood by researchers (Ref #6 Lipscomb 1990). Work within the past two decades has provided vital knowledge regarding the mechanisms of aminopeptidases. The mechanism of
bovine lens LAP and PepA have been elucidated (Ref 1 and 2), however, the exact mechanism of tomato LAP-A is unknown at this time. A search of current literature does not indicate that new research is underway to determine the exact mechanism of LAP-A. Based on the biochemical similarities of the LAPs between kingdoms, the mechanism of LAP-A may be similar to bovine lens LAP and PepA.
Biological Function of LAPs
- LAP-A
Once thought of as a housekeeping gene
Housekeeping gene
A housekeeping gene is typically a constitutive gene that is required for the maintenance of basic cellular function, and are found in all cells of an organism. Although some housekeeping genes are expressed at relatively constant levels , other housekeeping genes may vary depending on...
necessary only for protein turnover
Protein turnover
Protein turnover is the balance between protein synthesis and protein degradation. More synthesis than breakdown indicates an anabolic state that builds lean tissues, more breakdown than synthesis indicates a catabolic state that burns lean tissues....
, studies have demonstrated that LAP-A has a regulatory role in the immune response in tomato.
- Background On PlantPlantPlants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
Immune Response
In order to survive, plants must be able to respond to many biotic
Biotic component
Biotic components are the living things that shape an ecosystem. A biotic factor is any living component that affects another organism, including animals that consume the organism in question, and the living food that the organism consumes. Each biotic factor needs energy to do work and food for...
and abiotic stresses, including pathogen
Pathogen
A pathogen gignomai "I give birth to") or infectious agent — colloquially, a germ — is a microbe or microorganism such as a virus, bacterium, prion, or fungus that causes disease in its animal or plant host...
attack, piercing/sucking
Stylet
A stylet is a hard, sharp, anatomical structure found in some invertebrates.For example, the word stylet or stomatostyle, is used for the primitive piercing mouthparts of some nematodes and some nemerteans...
insect
Insect
Insects are a class of living creatures within the arthropods that have a chitinous exoskeleton, a three-part body , three pairs of jointed legs, compound eyes, and two antennae...
s, herbivory
Herbivore
Herbivores are organisms that are anatomically and physiologically adapted to eat plant-based foods. Herbivory is a form of consumption in which an organism principally eats autotrophs such as plants, algae and photosynthesizing bacteria. More generally, organisms that feed on autotrophs in...
, and mechanical wounding. These stresses activate specialized signal transduction pathways
Signal transduction
Signal transduction occurs when an extracellular signaling molecule activates a cell surface receptor. In turn, this receptor alters intracellular molecules creating a response...
, which are specific to the stressor and the amount of tissue damage inflicted. Similar to mechanical wounding, chewing insects, such as the tobacco hornworm (Manduca sexta, one of the major pests of tomato), cause extensive tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
damage activating the jasmonic acid
Jasmonic acid
Jasmonic acid is derived from the fatty acid linolenic acid. It is a member of the jasmonate class of plant hormones. It is biosynthesized from linolenic acid by the octadecanoid pathway....
(JA)-mediated response (Walling 2000). This JA-mediated response revolves around the octadecadnoid pathway, which is responsible for the synthesis of JA and several other potent signaling molecules, and ends in the regulation of two temporal
Temporal
Temporal can refer to:* of or relating to time** Temporality in philosophy** Temporal database, a database recording aspects of time varying values** The Temporal power of the Popes of the Roman Catholic Church...
ly distinct sets of genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
. The early genes amplify the wounding signal and can be detected 30 minutes to 2 hours after damage (Ryan 2000). Late gene expression can be seen 4–24 hours after wounding. Products of late-response genes act as deterrents to chewing-insect feeding, often by decreasing the nutritional value of the food ingested or interfering with insect gut function (Walling 2000). For example, serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
proteinase inhibitors (Pins) interfere with digestive proteases in the insect gut and polyphenol oxidase
Polyphenol oxidase
Polyphenol oxidase is a tetramer which contains four atoms of copper per molecule, and binding sites for two aromatic compounds and oxygen...
s (PPO) act to decrease the nutritive value of plant leaves after ingestion
Ingestion
Ingestion is the consumption of a substance by an organism. In animals, it normally is accomplished by taking in the substance through the mouth into the gastrointestinal tract, such as through eating or drinking...
by herbivores (Johnson et al. 1989; Ryan 2000; Orozco-Cardenas 2001). Please see the Picture 3 for a summary of the wound response in tomato.
The plant response in this octadecanoid pathway is similar to mammalian prostaglandin
Prostaglandin
A prostaglandin is any member of a group of lipid compounds that are derived enzymatically from fatty acids and have important functions in the animal body. Every prostaglandin contains 20 carbon atoms, including a 5-carbon ring....
and
leukotriene
Leukotriene
Leukotrienes are fatty signaling molecules. They were first found in leukocytes . One of their roles is to trigger contractions in the smooth muscles lining the trachea; their overproduction is a major cause of inflammation in asthma and allergic rhinitis...
pathways (Ref Walling 2000). This particular pathway is inhibited by salicylic acid
Salicylic acid
Salicylic acid is a monohydroxybenzoic acid, a type of phenolic acid and a beta hydroxy acid. This colorless crystalline organic acid is widely used in organic synthesis and functions as a plant hormone. It is derived from the metabolism of salicin...
.
- LAP-A and the Ocatadecanoid Pathway
(LAP-A), a product of the octadecanoid pathway in some solanaecous plants, has been shown by Fowler et al. to have a regulatory role in the late wound response of tomato. Experiments were conducted using three genotypes of tomato plants: wildtype (WT), (LapA-SI) plants that were silenced for LAP-A, and LapA-OX that constitutively expressed LAP-A. Late-gene expression was inhibited in wounded LapA-SI plants, and the LapA-SI plants were also more susceptible to tobacco hornworm feeding, relative to wildtype (WT) plants. In comparison, the wounded LapA-OX leaves exhibited heightened levels of late gene RNA accumulation, an increased resistance to herbivory, and extended expression of late wound-response genes. These data suggest that LAP-A functions in regulating both the intensity and the persistence of the late wound response. However, unwounded LapA-OX did not accumulate late gene RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
transcripts, suggesting that presence of LAP-A alone is not sufficient to induce late gene expression. LAP-A is the first plant aminopeptidase shown to have a regulatory role in signal transduction pathway.
External links
- The MEROPSMeropsMerops may refer to:* Merops , a genus of bee-eaters.* MEROPS, an on-line database for peptidases.It may also refer to several figures from Greek mythology:* King of Ethiopia, husband of Clymene, who lay with Helios and bore Phaethon...
online database for peptidases and their inhibitors: Animal:M17.001, Bacteria:M17.003, Plant::M17.002.