Leucyl aminopeptidase

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Leucine aminopeptidase
Crystal structure of bovine leucine aminopeptidase (PDB 1BLL).png
Crystal structure of bovine leucyl aminopeptidase with co-ordinated zinc ions. Rendered from PDB 1BLL.
Identifiers
SymbolLAP
Alt. symbolsPEPS
NCBI gene 51056
HGNC 18449
OMIM 170250
RefSeq NM_015907
UniProt P28838
Other data
EC number 3.4.11.1
Locus Chr. 4 p15.33
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Structures Swiss-model
Domains InterPro

Leucyl aminopeptidases (EC 3.4.11.1, leucine aminopeptidase, LAPs, leucyl peptidase, peptidase S, cytosol aminopeptidase, cathepsin III, L-leucine aminopeptidase, leucinaminopeptidase, leucinamide aminopeptidase, FTBL proteins, proteinates FTBL, aminopeptidase II, aminopeptidase III, aminopeptidase I) 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).

Contents

Enzyme description, structure, and active site

LAP-A active site residues. Two Zn+2 cations are also shown, along with a water and a bicarbonate ion that acts as a general base. Proposed LAP-A active site.png
LAP-A active site residues. Two Zn+2 cations are also shown, along with a water and a bicarbonate ion that acts as a general base.

The active sites in PepA and in bovine lens LAP have been found to be similar. [1] 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. [2] 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 [3] 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 60 °C. PepA, bovine lens LAP and LAP-A are also known to form hexamers in vivo . The Gu et al. from 1999 demonstrated that six 55kDA enzymatically inactive LAP-A protomers 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 [4] can be found through Protein Data Bank (2J9A).

Mechanism(s)

Historically, the mechanisms of carboxypeptidases 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

In this mechanism, the bicarbonate ion acts as a general base. For LAP-A, R1 could be the R group of leucine, methionine, or arginine. Hypothesized Mechanisms of Bovine lens LAP and PepA.png
In this mechanism, the bicarbonate ion acts as a general base. For LAP-A, R1 could be the R group of leucine, methionine, or arginine.

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

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.

Background on plant immune response

In order to survive, plants must be able to respond to many biotic and abiotic stresses, including pathogen attack, piercing/sucking insects, 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 octadecanoid pathway, which is responsible for the synthesis of JA and several other potent signaling molecules, and ends in the regulation of two sets of genes whose expression changes over time. 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 oxidases (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 wounding response pathway as studied in tomato. The Octadecanoid Pathway as studied in tomato.png
The wounding response pathway as studied 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.

Octadecanoid pathway

(LAP-A), a product of the octadecanoid pathway in some solanaceous 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.

Osmoregulation

LAP proteins are expressed in a variety of marine organisms as a method of coping with the osmotic threat high salinity poses to the cell. During bouts of high salinity, LAP begins the catalysis of proteins in order to release amino acids into the cell in an attempt to balance the high ion concentrations in the external environment. [5]

Related Research Articles

<span class="mw-page-title-main">Leucine</span> Chemical compound

Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α-amino group (which is in the protonated −NH3+ form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −COO form under biological conditions), and a side chain isobutyl group, making it a non-polar aliphatic amino acid. It is essential in humans, meaning the body cannot synthesize it: it must be obtained from the diet. Human dietary sources are foods that contain protein, such as meats, dairy products, soy products, and beans and other legumes. It is encoded by the codons UUA, UUG, CUU, CUC, CUA, and CUG.

<span class="mw-page-title-main">Jasmonate</span> Lipid-based plant hormones

Jasmonate (JA) and its derivatives are lipid-based plant hormones that regulate a wide range of processes in plants, ranging from growth and photosynthesis to reproductive development. In particular, JAs are critical for plant defense against herbivory and plant responses to poor environmental conditions and other kinds of abiotic and biotic challenges. Some JAs can also be released as volatile organic compounds (VOCs) to permit communication between plants in anticipation of mutual dangers.

<span class="mw-page-title-main">EGR1</span> Protein-coding gene in the species Homo sapiens

EGR-1 also known as ZNF268 or NGFI-A is a protein that in humans is encoded by the EGR1 gene.

<span class="mw-page-title-main">Leucine zipper</span> DNA-binding structural motif

A leucine zipper is a common three-dimensional structural motif in proteins. They were first described by Landschulz and collaborators in 1988 when they found that an enhancer binding protein had a very characteristic 30-amino acid segment and the display of these amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The polypeptide segments containing these periodic arrays of leucine residues were proposed to exist in an alpha-helical conformation and the leucine side chains from one alpha helix interdigitate with those from the alpha helix of a second polypeptide, facilitating dimerization.

A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence or have a general affinity to DNA. Some DNA-binding domains may also include nucleic acids in their folded structure.

<span class="mw-page-title-main">Adenine phosphoribosyltransferase</span> Mammalian protein found in Homo sapiens

Adenine phosphoribosyltransferase (APRTase) is an enzyme encoded by the APRT gene, found in humans on chromosome 16. It is part of the Type I PRTase family and is involved in the nucleotide salvage pathway, which provides an alternative to nucleotide biosynthesis de novo in humans and most other animals. In parasitic protozoa such as giardia, APRTase provides the sole mechanism by which AMP can be produced. APRTase deficiency contributes to the formation of kidney stones (urolithiasis) and to potential kidney failure.

<i>N</i>-Formylmethionine Chemical compound

N-Formylmethionine is a derivative of the amino acid methionine in which a formyl group has been added to the amino group. It is specifically used for initiation of protein synthesis from bacterial and organellar genes, and may be removed post-translationally.

<span class="mw-page-title-main">Systemin</span> Plant peptide hormone

Systemin is a plant peptide hormone involved in the wound response in the family Solanaceae. It was the first plant hormone that was proven to be a peptide having been isolated from tomato leaves in 1991 by a group led by Clarence A. Ryan. Since then, other peptides with similar functions have been identified in tomato and outside of the Solanaceae. Hydroxyproline-rich glycopeptides were found in tobacco in 2001 and AtPeps were found in Arabidopsis thaliana in 2006. Their precursors are found both in the cytoplasm and cell walls of plant cells, upon insect damage, the precursors are processed to produce one or more mature peptides. The receptor for systemin was first thought to be the same as the brassinolide receptor but this is now uncertain. The signal transduction processes that occur after the peptides bind are similar to the cytokine-mediated inflammatory immune response in animals. Early experiments showed that systemin travelled around the plant after insects had damaged the plant, activating systemic acquired resistance, now it is thought that it increases the production of jasmonic acid causing the same result. The main function of systemins is to coordinate defensive responses against insect herbivores but they also affect plant development. Systemin induces the production of protease inhibitors which protect against insect herbivores, other peptides activate defensins and modify root growth. They have also been shown to affect plants' responses to salt stress and UV radiation. AtPEPs have been shown to affect resistance against oomycetes and may allow A. thaliana to distinguish between different pathogens. In Nicotiana attenuata, some of the peptides have stopped being involved in defensive roles and instead affect flower morphology.

<span class="mw-page-title-main">Phosphoenolpyruvate carboxylase</span> Class of enzymes

Phosphoenolpyruvate carboxylase (also known as PEP carboxylase, PEPCase, or PEPC; EC 4.1.1.31, PDB ID: 3ZGE) is an enzyme in the family of carboxy-lyases found in plants and some bacteria that catalyzes the addition of bicarbonate (HCO3) to phosphoenolpyruvate (PEP) to form the four-carbon compound oxaloacetate and inorganic phosphate:

<span class="mw-page-title-main">Leucyl/cystinyl aminopeptidase</span> Protein-coding gene in the species Homo sapiens

Leucyl/cystinyl aminopeptidase, also known as cystinyl aminopeptidase (CAP), insulin-regulated aminopeptidase (IRAP), human placental leucine aminopeptidase (PLAP), oxytocinase, and vasopressinase, is an enzyme of the aminopeptidase group that in humans is encoded by the LNPEP gene.

<span class="mw-page-title-main">Enoyl-CoA hydratase</span>

Enoyl-CoA hydratase (ECH) or crotonase is an enzyme EC 4.2.1.17 that hydrates the double bond between the second and third carbons on 2-trans/cis-enoyl-CoA:

<span class="mw-page-title-main">12-oxophytodienoate reductase</span> Class of enzymes

12-oxophytodienoate reductase (OPRs) is an enzyme of the family of Old Yellow Enzymes (OYE). OPRs are grouped into two groups: OPRI and OPRII – the second group is the focus of this article, as the function of the first group is unknown, but is the subject of current research. The OPR enzyme utilizes the cofactor flavin mononucleotide (FMN) and catalyzes the following reaction in the jasmonic acid synthesis pathway:

<span class="mw-page-title-main">METAP2</span> Protein-coding gene in humans

Methionine aminopeptidase 2 is an enzyme that in humans is encoded by the METAP2 gene.

<span class="mw-page-title-main">Cutinase</span>

The enzyme cutinase is a member of the hydrolase family. It catalyzes the following reaction:

<span class="mw-page-title-main">ERAP1</span> Protein-coding gene in the species Homo sapiens

Type 1 tumor necrosis factor receptor shedding aminopeptidase regulator, also known as endoplasmic reticulum aminopeptidase 1 (ARTS-1), is a protein which in humans is encoded by the ARTS-1 gene.

<span class="mw-page-title-main">NPEPPS</span> Protein-coding gene in the species Homo sapiens

Puromycin-sensitive amino peptidase also known as cytosol alanyl aminopeptidase or alanine aminopeptidase (AAP) is an enzyme that in humans is encoded by the NPEPPS gene. It is used as a biomarker to detect damage to the kidneys, and that may be used to help diagnose certain kidney disorders. It is found at high levels in the urine when there are kidney problems.

The N-end rule is a rule that governs the rate of protein degradation through recognition of the N-terminal residue of proteins. The rule states that the N-terminal amino acid of a protein determines its half-life. The rule applies to both eukaryotic and prokaryotic organisms, but with different strength, rules, and outcome. In eukaryotic cells, these N-terminal residues are recognized and targeted by ubiquitin ligases, mediating ubiquitination thereby marking the protein for degradation. The rule was initially discovered by Alexander Varshavsky and co-workers in 1986. However, only rough estimations of protein half-life can be deduced from this 'rule', as N-terminal amino acid modification can lead to variability and anomalies, whilst amino acid impact can also change from organism to organism. Other degradation signals, known as degrons, can also be found in sequence.

<span class="mw-page-title-main">Polygalacturonase</span>

Endo-polygalacturonase (EC 3.2.1.15, pectin depolymerase, pectolase, pectin hydrolase, and poly-α-1,4-galacturonide glycanohydrolase; systematic name (1→4)-α-D-galacturonan glycanohydrolase (endo-cleaving)) is an enzyme that hydrolyzes the α-1,4 glycosidic bonds between galacturonic acid residues:

<span class="mw-page-title-main">Leucine-rich repeat receptor like protein kinase</span>

Leucine-rich repeat receptor like protein kinase are plant cell membrane localized Leucine-rich repeat (LRR) receptor kinase that play critical roles in plant innate immunity. Plants have evolved intricate immunity mechanism to combat against pathogen infection by recognizing Pathogen Associated Molecular Patterns (PAMP) and endogenous Damage Associated Molecular Patterns (DAMP). PEPR 1 considered as the first known DAMP receptor of Arabidopsis.

<span class="mw-page-title-main">Arginylation</span> Arginylation Post-translational modification

Arginylation is a post-translational modification in which proteins are modified by the addition of arginine (Arg) at the N-terminal amino group or side chains of reactive amino acids by the enzyme, arginyltransferase (ATE1). Recent studies have also revealed that hundreds of proteins in vivo are arginylated, proteins which are essential for many biological pathways. While still poorly understood in a biological setting, the ATE1 enzyme is highly conserved which suggests that arginylation is an important biological post-translational modification.

References

  1. Sträter N, Sun L, Kantrowitz ER, Lipscomb WN (September 1999). "A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase". Proceedings of the National Academy of Sciences of the United States of America . 96 (20): 11151–5. Bibcode:1999PNAS...9611151S. doi: 10.1073/pnas.96.20.11151 . PMC   18002 . PMID   10500145.
  2. Gu YQ, Walling LL (March 2002). "Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato". European Journal of Biochemistry . 269 (6): 1630–40. doi: 10.1046/j.1432-1327.2002.02795.x . PMID   11895433.
  3. Gu YQ, Holzer FM, Walling LL (August 1999). "Overexpression, purification and biochemical characterization of the wound-induced leucine aminopeptidase of tomato". European Journal of Biochemistry . 263 (3): 726–35. doi: 10.1046/j.1432-1327.1999.00548.x . PMID   10469136.
  4. Kraft M, Schleberger C, Weckesser J, Schulz GE (December 2006). "Binding structure of the leucine aminopeptidase inhibitor microginin FR1". FEBS Letters . 580 (30): 6943–7. doi: 10.1016/j.febslet.2006.11.060 . PMID   17157838. S2CID   6425967.
  5. Hilbish TJ (1985). "The Physiological Basis of Natural Selection at the Lap Locus". Evolution. 39 (6): 1302–1317. doi:10.2307/2408787. JSTOR   2408787. PMID   28564261.
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