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User:Dinosome/Cutinase

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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:

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.

Enzymatic Nomenclature

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Carboxylic ester structure
Carboxylic ester structure that is targeted by enzymes in the first subclass of class 3 enzymes.

Cutinase has an assigned enzyme commission number of EC 3.1.1.74.[1] Cutinase is in the third class of enzymes, meaning that its primary function is to hydrolyze its substrate (in this case, cutin).[2] Within the third class, cutinase is further categorized into the first subclass, which indicates that it specifically hydrolyzes ester bonds.[1] It is then placed in the first sub-subclass, meaning that it targets carboxylic esters, which are those that join together cutin polymers.[1]

Functions in Organisms

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Cutin layers on a plant cell
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.[3] This layer of cutin is formed by a matrix-like structure that contains waxy components embedded in the carbohydrate layers.[4] 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.[3][5]

Research suggests that cutin plays a critical role in preventing pathogenic infections in plant systems.[6] 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.[7]

Cutinase is produced by a variety of fungal plant pathogens, and its activity was first detected in the fungus, Penicillium spinulosum.[8] 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.[8] 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.[8] 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.[8][9] Inhibition of cutinase has been shown to prevent fungal infection through intact cuticles.[9] Conversely, the supplementation of cutinase to fungi that are not able to produce it naturally had been shown to enhance fungal infection success rates.[8]

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.[10][11] 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.[10]

Crystal Structure and Insights

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Cutinase belongs to the α-β class of proteins, with a central β-sheet of 5 parallel strands covered by 5 helices on either side of the sheet.[12] Fungal cutinase is generally composed of around 197 amino acid residues, and its native form consists of a single domain.[13] The protein also contains 4 invariant cysteine residues that form 2 disulfide bridges, whose cleavage results in a complete loss of enzymatic activity.[14][13]

Crystal structures have shown that the active site of cutinases is found on one end of the ellipsoid shape of the enzyme.[15] This active site is seen flanked by two hydrophobic loop structures and partly covered by 2 thin bridges formed by amino acid side chains.[12][15] It does not possess a hydrophobic lid, which is a common constituent feature among other lipases.[12] 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.[12][13] Cutinase activation is believed to be derived from slight shifts in the conformation of hydrophobic residues, acting as a miniature lid.[12] 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.[16]

Structure-Function Mechanism

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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.[14][17] 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.[17] These hydrophobic residues show a higher degree of flexibility, suggesting an induced fit model to facilitate cutin bonding to the active site.[12] In the cutinase active site, histidine deprotonates serine, allowing the serine to undergo a nucleophilic attack on the cutin carboxylic ester.[18] 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.[18] The process repeats with a nucleophilic attack on the new carboxylic ester by a deprotonated water molecule.[18] Following this, the charged oxygen reforms its double bond, removing the serine attachment and releasing the carboxylic acid R monomer.[18]

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.[18]

Industrial Applications

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The stability of cutinases in higher temperatures (20-50°C) and its compatibility with other hydrolytic enzymes has potential applications in the detergent industry.[19] 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.[20] 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.[19] 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.[19]

Rather unexpectedly, the ability to degrade the cutin layer of plants and their fruits holds the potential to be beneficial to the fruit industry.[19] This is because the cuticle layer of fruits is a putative mechanism of water regulation, and the degradation of this layer subject the fruits to water movement across its membrane.[21] 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.[19]

References

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  1. ^ a b c "IUBMB Nomenclature Home Page". web.archive.org. 2018-10-10. Retrieved 2022-09-29.
  2. ^ McDonald, Andrew G.; Tipton, Keith F. (2022-01-03). "Enzyme nomenclature and classification: the state of the art". The FEBS Journal: febs.16274. doi:10.1111/febs.16274. ISSN 1742-464X.
  3. ^ a b Heredia, Antonio (2003-03). "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. ISSN 0304-4165. {{cite journal}}: Check date values in: |date= (help)
  4. ^ Walton, T. J. (1990). "Waxes, cutin and suberin". Methods Plant Biochemisry. 4: 105–158.
  5. ^ G., Kerstiens, (1996). Plant cuticles : an integrated functional approach. BIOS Scientific Publishers. ISBN 1-85996-130-4. OCLC 36076660.{{cite book}}: CS1 maint: extra punctuation (link) CS1 maint: multiple names: authors list (link)
  6. ^ Fich, Eric A.; Segerson, Nicholas A.; Rose, Jocelyn K.C. (2016-04-29). "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. ISSN 1543-5008.
  7. ^ Fernie, Alisdair; Nunes-Nesi, Adriano (2009-09-21). "Faculty Opinions recommendation of Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss". Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature. Retrieved 2022-09-30.
  8. ^ a b c d e Schäfer, Wilhelm (1993-05). "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. {{cite journal}}: Check date values in: |date= (help)
  9. ^ a b Sweigard, James A.; Chumley, Forrest G.; Valent, Barbara (1992-03). "Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea". Molecular and General Genetics MGG. 232 (2): 174–182. doi:10.1007/bf00279994. ISSN 0026-8925. {{cite journal}}: Check date values in: |date= (help)
  10. ^ a b Chen, Sheng; Tong, Xing; Woodard, Ronald W.; Du, Guocheng; Wu, Jing; Chen, Jian (2008-09). "Identification and Characterization of Bacterial Cutinase". Journal of Biological Chemistry. 283 (38): 25854–25862. doi:10.1074/jbc.m800848200. ISSN 0021-9258. {{cite journal}}: Check date values in: |date= (help)CS1 maint: unflagged free DOI (link)
  11. ^ Fett, W. F.; Wijey, C.; Moreau, R. A.; Osman, S. F. (1999-04). "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. {{cite journal}}: Check date values in: |date= (help)
  12. ^ a b c d e f Martinez, C.; Cambillau, C. (1994-07-31). "FUSARIUM SOLANI CUTINASE IS A LIPOLYTIC ENZYME WITH A CATALYTIC SERINE ACCESSIBLE TO SOLVENT". dx.doi.org. Retrieved 2022-09-28.
  13. ^ a b c Carvalho, Cristina M. L.; Aires-Barros, Maria Raquel; Cabral, Joaquim M. S. (1998-12-15). "Cutinase structure, function and biocatalytic applications". Electronic Journal of Biotechnology. 1 (2): 160–173. doi:10.2225/vol1-issue3-fulltext-8. ISSN 0717-3458.
  14. ^ a b Ettinger, William F.; Thukral, Sushil K.; Kolattukudy, Pappachan E. (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.
  15. ^ a b Jelsch, Christian; Longhi, Sonia; Cambillau, Christian (1998-05-15). <320::aid-prot8>3.0.co;2-m "Packing forces in nine crystal forms of cutinase". Proteins: Structure, Function, and Genetics. 31 (3): 320–333. doi:10.1002/(sici)1097-0134(19980515)31:3<320::aid-prot8>3.0.co;2-m. ISSN 0887-3585.
  16. ^ Cambillau, C.; Martinez, C.; Nicolas, A. (1996-03-08). "CONTRIBUTION OF CUTINASE SERINE 42 SIDE CHAIN TO THE STABILIZATION OF THE OXYANION TRANSITION STATE". dx.doi.org. Retrieved 2022-09-28.
  17. ^ a b Martinez, C.; Cambillau, C. (1994-08-31). "CUTINASE, A LIPOLYTIC ENZYME WITH A PREFORMED OXYANION HOLE". dx.doi.org. Retrieved 2022-09-28.
  18. ^ a b c d e Leung, Mei; Holliday, Gemma; Willey, James. "Cutinase". Mechanism and Catalytic Site Atlas. Retrieved 2022-09-28.
  19. ^ a b c d e Dutta, Kasturi; Sen, Shampa; Veeranki, Venkata Dasu (2009-02). "Production, characterization and applications of microbial cutinases". Process Biochemistry. 44 (2): 127–134. doi:10.1016/j.procbio.2008.09.008. ISSN 1359-5113. {{cite journal}}: Check date values in: |date= (help)
  20. ^ Egmond, Maarten R.; van Bemmel, Carla J. (1997), "[6] Impact of structural information on understanding lipolytic function", Methods in Enzymology, Elsevier, pp. 119–129, retrieved 2022-09-30
  21. ^ "5510131 Enzyme assisted degradation of surface membranes of harvested fruits and vegetables". Biotechnology Advances. 15 (1): 273. 1997-01. doi:10.1016/s0734-9750(97)88551-5. ISSN 0734-9750. {{cite journal}}: Check date values in: |date= (help)
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