Cutinase

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cutinase
PDB 1cex EBI.jpg
Structure of Fusarium solani cutinase. PDB 1cex . [1]
Identifiers
EC no. 3.1.1.74
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The enzyme cutinase (systematic name: cutin hydrolase, EC 3.1.1.74) is a member of the hydrolase family. It catalyzes the following reaction:

Contents

Cutinase
Identifiers
SymbolCutinase
Pfam PF01083
InterPro IPR000675
PROSITE PDOC00140
SCOP2 1cex / SCOPe / SUPFAM
OPM superfamily 127
OPM protein 1oxm
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary

In biological systems, the reactant carboxylic ester is a constituent of the cutin polymer, and the hydrolysis of cutin results in the formation of alcohol and carboxylic acid monomer products.

Nomenclature

Cutinase has an assigned enzyme commission number of EC 3.1.1.74. [2] Cutinase is in the third class of enzymes, meaning that its primary function is to hydrolyze its substrate (in this case, cutin). [3] Within the third class, cutinase is further categorized into the first subclass, which indicates that it specifically hydrolyzes ester bonds. [2] It is then placed in the first sub-subclass, meaning that it targets carboxylic esters, which are those that join together cutin polymers. [2]

Function

Cutin composes most of the waxy cuticle layer around plant cells. In order to enter plant cells, pathogens need to traverse this barrier. Cuticle overlying upper epidermis in mesophyte leaf (35103215772).jpg
Cutin composes most of the waxy cuticle layer around plant cells. In order to enter plant cells, pathogens need to traverse this barrier.

Most plants have a layer composed of cutin, called the cuticle, on their aboveground surfaces such as stems, leaves, and fruits. [4] This layer of cutin is formed by a matrix-like structure that contains waxy components embedded in the carbohydrate layers. [5] The molecule, cutin, which composes most of the cuticle matrix (40-80%), is composed primarily of fatty acid chains that are polymerized via carboxylic ester bonds. [4] [6]

Research suggests that cutin plays a critical role in preventing pathogenic infections in plant systems. [7] For instance, experiments conducted on tomato plants that had a substantial inability to synthesize cutin found that that tomatoes produced by those plants were significantly more susceptible to infection by both opportunistic pathogens and intentionally inoculated fungal spores. [8]

Cutinase is produced by a variety of fungal plant pathogens, and its activity was first detected in the fungus, Penicillium spinulosum. [9] In studies of Nectria haematococca, a fungal pathogen that is the cause of foot rot in pea plants, cutinase has been shown to play key roles in facilitating the early stages of plant infection. [9] It is also suggested that fungal spores that make initial contact with plant surfaces, a small amount of catalytic cutinase produces cutin monomers which in turn up-regulate the expression of the cutinase gene. [9] This proposes that the expression pathway of cutinase in fungal spores is characterized by a positive feedback loop until the fungus successfully breaches the cutin layer; however, the specific mechanism of this pathway is unclear. [9] [10] Inhibition of cutinase has been shown to prevent fungal infection through intact cuticles. [10] Conversely, the supplementation of cutinase to fungi that are not able to produce it naturally had been shown to enhance fungal infection success rates. [9]

Cutinases have also been observed in a few plant pathogenic bacterial species, such as Streptomyces scabies, Thermobifida fusca, Pseudomonas mendocina, and Pseudomonas putida, but these have not been studied to the extent as those found in fungi. [11] [12] The molecular structure of the Thermobifida fusca cutinase shows similarities to the Fusarium solani pisi fungal cutinase, with congruencies in their active sites and overall mechanisms. [11]

Structure

Cutinase belongs to the α-β class of proteins, with a central β-sheet of 5 parallel strands covered by 5 alpha helices on either side of the sheet. [13] Fungal cutinase is generally composed of around 197 amino acid residues, and its native form consists of a single domain. [14] The protein also contains 4 invariant cysteine residues that form 2 disulfide bridges, whose cleavage results in a complete loss of enzymatic activity. [15] [14]

Crystal structures have shown that the active site of cutinases is found on one end of the ellipsoid shape of the enzyme. [16] This active site is seen flanked by two hydrophobic loop structures and partly covered by 2 thin bridges formed by amino acid side chains. [13] [16] It does not possess a hydrophobic lid, which is a common constituent feature among other lipases. [13] Instead, the catalytic serine in the active site is exposed to open solvent, and the cutinase enzyme does not show interfacial activation behaviors at an aqueous-nonpolar interface. [13] [14] Cutinase activation is believed to be derived from slight shifts in the conformation of hydrophobic residues, acting as a miniature lid. [13] The oxyanion hole in the active site is a constituent feature of the binding site, which differs from most lipolytic enzymes whose oxyanion holes are induced upon substrate binding. [17]

Mechanism

Cutinase is a serine esterase, and the active site contains a serine-histidine-aspartate triad and an oxyanion hole, which are signature elements of serine hydrolases. [15] [18] The binding site of the cutin lipid polymer consists of two hydrophobic loops characterized by nonpolar amino acids such as leucine, alanine, isoleucine, and proline. [18] These hydrophobic residues show a higher degree of flexibility, suggesting an induced fit model to facilitate cutin bonding to the active site. [13] In the cutinase active site, histidine deprotonates serine, allowing the serine to undergo a nucleophilic attack on the cutin carboxylic ester. [19] This is followed by an elimination reaction whereby the charged oxygen (stabilized by the oxyanion hole) creates a double bond, removing an R group from the cutin polymer in the form of an alcohol. [19] The process repeats with a nucleophilic attack on the new carboxylic ester by a deprotonated water molecule. [19] Following this, the charged oxygen reforms its double bond, removing the serine attachment and releasing the carboxylic acid R monomer. [19]

Step by step mechanism of the hydrolysis of cutin polymers via the serine-histidine-aspartate residues in the active site of cutinase. Image adapted from Mei Leung, Gemma L. Holliday, and James Willey. Cutinase Hydrolysis Mechanism.png
Step by step mechanism of the hydrolysis of cutin polymers via the serine-histidine-aspartate residues in the active site of cutinase. Image adapted from Mei Leung, Gemma L. Holliday, and James Willey.

Applications

The stability of cutinases in higher temperatures (20-50 °C) and its compatibility with other hydrolytic enzymes has potential applications in the detergent industry. [20] In fact, it has been shown that cutinases are more efficient at cleaving and eliminating non-calcium fats from clothing when compared against other industrial lipases. [21] Another advantage of cutinase in this industry is its ability to be catalytically active with both water- and lipid-soluble ester compounds, making it a more versatile degradative agent. [20] This versatility is also subjecting cutinase to experiments in enhancing the biofuel industry because of its ability to facilitate transesterification of biofuels in various solubility environments. [20]

Rather unexpectedly, the ability to degrade the cutin layer of plants and their fruits holds the potential to be beneficial to the fruit industry. [20] This is because the cuticle layer of fruits is a putative mechanism of water regulation, and the degradation of this layer subjects the fruits to water movement across its membrane. [22] By using cutinase to degrade the cuticle of fruits, industry makers can enhance the drying of fruits and more easily deliver preservatives and additives to the flesh of the fruit. [20]

See also

Related Research Articles

<span class="mw-page-title-main">Amino acid</span> Organic compounds containing amine and carboxylic groups

Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 appear in the genetic code of life.

<span class="mw-page-title-main">Chymotrypsin</span> Digestive enzyme

Chymotrypsin (EC 3.4.21.1, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the S1 position) of the enzyme. It is activated in the presence of trypsin. The hydrophobic and shape complementarity between the peptide substrate P1 side chain and the enzyme S1 binding cavity accounts for the substrate specificity of this enzyme. Chymotrypsin also hydrolyzes other amide bonds in peptides at slower rates, particularly those containing leucine at the P1 position.

<span class="mw-page-title-main">Serine protease</span> Class of enzymes

Serine proteases are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

A metalloproteinase, or metalloprotease, is any protease enzyme whose catalytic mechanism involves a metal. An example is ADAM12 which plays a significant role in the fusion of muscle cells during embryo development, in a process known as myogenesis.

<span class="mw-page-title-main">DD-transpeptidase</span> Bacterial enzyme

DD-transpeptidase is a bacterial enzyme that catalyzes the transfer of the R-L-αα-D-alanyl moiety of R-L-αα-D-alanyl-D-alanine carbonyl donors to the γ-OH of their active-site serine and from this to a final acceptor. It is involved in bacterial cell wall biosynthesis, namely, the transpeptidation that crosslinks the peptide side chains of peptidoglycan strands.

Cutin is one of two waxy polymers that are the main components of the plant cuticle, which covers all aerial surfaces of plants, the other being cutan. It is an insoluble substance with waterproof quality. Cutin also harbors cuticular waxes, which assist in cuticle structure. Cutan, the other major cuticle polymer, is much more readily preserved in fossil records. Cutin consists of omega hydroxy acids and their derivatives, which are interlinked via ester bonds, forming a polyester polymer of indeterminate size.

<span class="mw-page-title-main">Appressorium</span> Structure produced by some fungi

An appressorium is a specialized cell typical of many fungal plant pathogens that is used to infect host plants. It is a flattened, hyphal "pressing" organ, from which a minute infection peg grows and enters the host, using turgor pressure capable of punching through even Mylar.

<span class="mw-page-title-main">Polyester</span> Category of polymers, in which the monomers are joined together by ester links

Polyester is a category of polymers that contain the ester functional group in every repeat unit of their main chain. As a specific material, it most commonly refers to a type called polyethylene terephthalate (PET). Polyesters include naturally occurring chemicals, such as in plants and insects, as well as synthetics such as polybutyrate. Natural polyesters and a few synthetic ones are biodegradable, but most synthetic polyesters are not. Synthetic polyesters are used extensively in clothing.

<span class="mw-page-title-main">Cysteine protease</span> Class of enzymes

Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.

<span class="mw-page-title-main">Plant cuticle</span> Waterproof covering of aerial plant organs

A plant cuticle is a protecting film covering the outermost skin layer (epidermis) of leaves, young shoots and other aerial plant organs that have no periderm. The film consists of lipid and hydrocarbon polymers infused with wax, and is synthesized exclusively by the epidermal cells.

<span class="mw-page-title-main">Diacylglycerol lipase</span> Enzyme that breaks down diacylglycerol in many organisms.

Diacylglycerol lipase, also known as DAG lipase, DAGL, or DGL, is an enzyme that catalyzes the hydrolysis of diacylglycerol, releasing a free fatty acid and monoacylglycerol:

diacylglycerol + H2O ⇌ monoacylglycerol + free fatty acid

<span class="mw-page-title-main">Chlorophyllase</span> Enzyme in chlorophyll metabolism

Chlorophyllase is an essential enzyme in chlorophyll metabolism. It is a membrane proteins commonly known as chlase (EC 3.1.1.14, CLH) with systematic name chlorophyll chlorophyllidohydrolase. It catalyzes the reaction

<span class="mw-page-title-main">Gastric lipase</span> Class of enzymes

Gastric lipase, also known as LIPF, is an enzymatic protein that, in humans, is encoded by the LIPF gene.

<span class="mw-page-title-main">Steroid Delta-isomerase</span>

In enzymology, a steroid Δ5-isomerase is an enzyme that catalyzes the chemical reaction

The enzyme carboxylesterase (or carboxylic-ester hydrolase, EC 3.1.1.1; systematic name carboxylic-ester hydrolase) catalyzes reactions of the following form:

<span class="mw-page-title-main">KLK6</span> Protein-coding gene in the species Homo sapiens

Kallikrein-6 is a protein that in humans is encoded by the KLK6 gene. Kallikrein-6 is also referred to as neurosin, protease M, hK6, or zyme. It is a 223 amino acid sequence, derived from its 244 original form, which contains a 16 residue presignal and 5 residue activation peptide.

<span class="mw-page-title-main">Lipase</span> Class of enzymes which cleave fats via hydrolysis

In biochemistry, lipase refers to a class of enzymes that catalyzes the hydrolysis of fats. Some lipases display broad substrate scope including esters of cholesterol, phospholipids, and of lipid-soluble vitamins and sphingomyelinases; however, these are usually treated separately from "conventional" lipases. Unlike esterases, which function in water, lipases "are activated only when adsorbed to an oil–water interface". Lipases perform essential roles in digestion, transport and processing of dietary lipids in most, if not all, organisms.

Omega hydroxy acids are a class of naturally occurring straight-chain aliphatic organic acids n carbon atoms long with a carboxyl group at position 1, and a hydroxyl at terminal position n where n > 3. They are a subclass of hydroxycarboxylic acids. The C16 and C18 omega hydroxy acids 16-hydroxy palmitic acid and 18-hydroxy stearic acid are key monomers of cutin in the plant cuticle. The polymer cutin is formed by interesterification of omega hydroxy acids and derivatives of them that are substituted in mid-chain, such as 10,16-dihydroxy palmitic acid. Only the epidermal cells of plants synthesize cutin.

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

Subtilases are a family of subtilisin-like serine proteases. They appear to have independently and convergently evolved an Asp/Ser/His catalytic triad, like in the trypsin serine proteases. The structure of proteins in this family shows that they have an alpha/beta fold containing a 7-stranded parallel beta sheet.

<span class="mw-page-title-main">Discovery and development of gastrointestinal lipase inhibitors</span>

Lipase inhibitors belong to a drug class that is used as an antiobesity agent. Their mode of action is to inhibit gastric and pancreatic lipases, enzymes that play an important role in the digestion of dietary fat. Lipase inhibitors are classified in the ATC-classification system as A08AB . Numerous compounds have been either isolated from nature, semi-synthesized, or fully synthesized and then screened for their lipase inhibitory activity but the only lipase inhibitor on the market is orlistat . Lipase inhibitors have also shown anticancer activity, by inhibiting fatty acid synthase.

References

  1. ↑ Longhi S, Czjzek M, Lamzin V, Nicolas A, Cambillau C (May 1997). "Atomic resolution (1.0 A) crystal structure of Fusarium solani cutinase: stereochemical analysis". Journal of Molecular Biology. 268 (4): 779–799. doi:10.1006/jmbi.1997.1000. PMID   9175860.
  2. 1 2 3 "EC 3.1.1.74: Cutinase". IUBMB Nomenclature Home Page. 2018-10-10. Archived from the original on 2018-10-04. Retrieved 2022-09-29.
  3. ↑ McDonald AG, Tipton KF (November 2021). "Enzyme nomenclature and classification: the state of the art". The FEBS Journal. 290 (9): 2214–2231. doi: 10.1111/febs.16274 . PMID   34773359. S2CID   244076461.
  4. 1 2 Heredia A (March 2003). "Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer". Biochimica et Biophysica Acta (BBA) - General Subjects. 1620 (1–3): 1–7. doi:10.1016/s0304-4165(02)00510-x. PMID   12595066.
  5. ↑ Walton TJ (1990). "Waxes, cutin and suberin". Methods Plant Biochemisry. 4: 105–158.
  6. ↑ Kerstiens G (1996). Plant cuticles : an integrated functional approach. BIOS Scientific Publishers. ISBN   1-85996-130-4. OCLC   36076660.
  7. ↑ Fich EA, Segerson NA, Rose JK (April 2016). "The Plant Polyester Cutin: Biosynthesis, Structure, and Biological Roles". Annual Review of Plant Biology. 67 (1): 207–233. doi:10.1146/annurev-arplant-043015-111929. PMID   26865339.
  8. ↑ Isaacson T, Kosma DK, Matas AJ, Buda GJ, He Y, Yu B, et al. (October 2009). "Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss". The Plant Journal. 60 (2): 363–377. doi: 10.1111/j.1365-313X.2009.03969.x . PMID   19594708.; Lay summary in: Fernie A, Nunes-Nesi A. "Faculty Opinions". Post-Publication Peer Review of the Biomedical Literature. doi: 10.3410/f.1164954.625804 .
  9. 1 2 3 4 5 Schäfer W (May 1993). "The role of cutinase in fungal pathogenicity". Trends in Microbiology. 1 (2): 69–71. doi:10.1016/0966-842x(93)90037-r. ISSN   0966-842X. PMID   8044466.
  10. 1 2 Sweigard JA, Chumley FG, Valent B (March 1992). "Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea". Molecular & General Genetics. 232 (2): 174–182. doi:10.1007/bf00279994. PMID   1557023. S2CID   37444.
  11. 1 2 Chen S, Tong X, Woodard RW, Du G, Wu J, Chen J (September 2008). "Identification and characterization of bacterial cutinase". The Journal of Biological Chemistry. 283 (38): 25854–25862. doi: 10.1074/jbc.m800848200 . PMC   3258855 . PMID   18658138.
  12. ↑ Fett WF, Wijey C, Moreau RA, Osman SF (April 1999). "Production of cutinase byThermomonospora fuscaATCC 27730". Journal of Applied Microbiology. 86 (4): 561–568. doi:10.1046/j.1365-2672.1999.00690.x. ISSN   1364-5072. S2CID   41512145.
  13. 1 2 3 4 5 6 Martinez C, De Geus P, Lauwereys M, Matthyssens G, Cambillau C (April 1992). "Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent". Nature. 356 (6370): 615–618. Bibcode:1992Natur.356..615M. doi:10.1038/356615a0. PMID   1560844. S2CID   4334360.
  14. 1 2 3 Carvalho CM, Aires-Barros MR, Cabral J (1998-12-15). "Cutinase structure, function and biocatalytic applications". Electronic Journal of Biotechnology. 1 (2): 160–173. doi:10.2225/vol1-issue3-fulltext-8. hdl: 1807/2161 . ISSN   0717-3458.
  15. 1 2 Ettinger WF, Thukral SK, Kolattukudy PE (1987-12-01). "Structure of cutinase gene, cDNA, and the derived amino acid sequence from phytopathogenic fungi". Biochemistry. 26 (24): 7883–7892. doi:10.1021/bi00398a052. ISSN   0006-2960.
  16. 1 2 Jelsch C, Longhi S, Cambillau C (May 1998). "Packing forces in nine crystal forms of cutinase". Proteins. 31 (3): 320–333. doi:10.1002/(sici)1097-0134(19980515)31:3<320::aid-prot8>3.0.co;2-m. PMID   9593202. S2CID   29668663.
  17. ↑ Nicolas A, Egmond M, Verrips CT, de Vlieg J, Longhi S, Cambillau C, Martinez C (January 1996). "Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state". Biochemistry. 35 (2): 398–410. doi:10.1021/bi9515578. PMID   8555209.
  18. 1 2 Martinez C, Nicolas A, van Tilbeurgh H, Egloff MP, Cudrey C, Verger R, Cambillau C (January 1994). "Cutinase, a lipolytic enzyme with a preformed oxyanion hole". Biochemistry. 33 (1): 83–89. doi:10.1021/bi00167a011. PMID   8286366.
  19. 1 2 3 4 Leung M, Holliday G, Willey J. "Cutinase". Mechanism and Catalytic Site Atlas. Retrieved 2022-09-28.
  20. 1 2 3 4 5 Dutta K, Sen S, Veeranki VD (February 2009). "Production, characterization and applications of microbial cutinases". Process Biochemistry. 44 (2): 127–134. doi:10.1016/j.procbio.2008.09.008. ISSN   1359-5113.
  21. ↑ Egmond MR, van Bemmel CJ (1997). "[6] Impact of structural information on understanding lipolytic function". Impact of structural information on understanding lipolytic function. Methods in Enzymology. Vol. 284. Elsevier. pp. 119–129. doi:10.1016/s0076-6879(97)84008-6. ISBN   9780121821852. PMID   9379930.
  22. ↑ "5510131 Enzyme assisted degradation of surface membranes of harvested fruits and vegetables". Biotechnology Advances. 15 (1): 273. January 1997. doi:10.1016/s0734-9750(97)88551-5. ISSN   0734-9750.

Further reading

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