Cutinase

Cutinase has an assigned enzyme commission number of EC 3.1.1.74.{{Cite web |date=2018-10-10 | title = EC 3.1.1.74: Cutinase | work = IUBMB Nomenclature Home Page |url=http://www.sbcs.qmul.ac.uk/iubmb/enzyme/EC3/1/1/74.html |access-date=2022-09-29 |archive-url=https://web.archive.org/web/20181004031030/http://www.sbcs.qmul.ac.uk/iubmb/enzyme/EC3/1/1/74.html |archive-date=2018-10-04 }} Cutinase is in the third class of enzymes, meaning that its primary function is to hydrolyze its substrate (in this case, cutin).{{cite journal | vauthors = McDonald AG, Tipton KF | title = Enzyme nomenclature and classification: the state of the art | journal = The FEBS Journal | pages = 2214–2231 | date = November 2021 | volume = 290 | issue = 9 | pmid = 34773359 | doi = 10.1111/febs.16274 | s2cid = 244076461 | doi-access = free }} Within the third class, cutinase is further categorized into the first subclass, which indicates that it specifically hydrolyzes ester bonds. It is then placed in the first sub-subclass, meaning that it targets carboxylic esters, which are those that join together cutin polymers.

File:Cuticle overlying upper epidermis in mesophyte leaf (35103215772).jpgMost plants have a layer composed of cutin, called the cuticle, on their aboveground surfaces such as stems, leaves, and fruits.{{cite journal | vauthors = Heredia A | title = Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer | journal = Biochimica et Biophysica Acta (BBA) - General Subjects | volume = 1620 | issue = 1–3 | pages = 1–7 | date = March 2003 | pmid = 12595066 | doi = 10.1016/s0304-4165(02)00510-x }} This layer of cutin is formed by a matrix-like structure that contains waxy components embedded in the carbohydrate layers.{{Cite journal | vauthors = Walton TJ |date=1990 |title=Waxes, cutin and suberin |url=https://www.academia.edu/24494725 |journal=Methods Plant Biochemisry |volume=4 |pages=105–158}} 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.{{Cite book | vauthors = Kerstiens G |url= http://worldcat.org/oclc/36076660 |title=Plant cuticles : an integrated functional approach |date=1996 |publisher=BIOS Scientific Publishers |isbn=1-85996-130-4 |oclc=36076660}}

Research suggests that cutin plays a critical role in preventing pathogenic infections in plant systems.{{cite journal | vauthors = Fich EA, Segerson NA, Rose JK | title = The Plant Polyester Cutin: Biosynthesis, Structure, and Biological Roles | journal = Annual Review of Plant Biology | volume = 67 | issue = 1 | pages = 207–233 | date = April 2016 | pmid = 26865339 | doi = 10.1146/annurev-arplant-043015-111929 }} For instance, experiments conducted on tomato plants that had a substantial inability to synthesize cutin found that the tomatoes produced by those plants were significantly more susceptible to infection by both opportunistic pathogens and intentionally inoculated fungal spores.{{cite journal | vauthors = Isaacson T, Kosma DK, Matas AJ, Buda GJ, He Y, Yu B, Pravitasari A, Batteas JD, Stark RE, Jenks MA, Rose JK | display-authors = 6 | title = Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss | journal = The Plant Journal | volume = 60 | issue = 2 | pages = 363–377 | date = October 2009 | pmid = 19594708 | doi = 10.1111/j.1365-313X.2009.03969.x | doi-access = free }}; {{lay source |template = journal | vauthors = Fernie A, Nunes-Nesi A | title = Faculty Opinions | journal = Post-Publication Peer Review of the Biomedical Literature | doi = 10.3410/f.1164954.625804 | url = https://facultyopinions.com/article/1164954#related-articles |doi-access=free}}

Cutinase is produced by a variety of fungal plant pathogens, and its activity was first detected in the fungus, Penicillium spinulosum.{{Cite journal | vauthors = Schäfer W |date=May 1993 |title=The role of cutinase in fungal pathogenicity |journal=Trends in Microbiology |volume=1 |issue=2 |pages=69–71 |doi=10.1016/0966-842x(93)90037-r |pmid=8044466 |issn=0966-842X}} 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. 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. 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.{{cite journal | vauthors = Sweigard JA, Chumley FG, Valent B | title = Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea | journal = Molecular & General Genetics | volume = 232 | issue = 2 | pages = 174–182 | date = March 1992 | pmid = 1557023 | doi = 10.1007/bf00279994 | s2cid = 37444 }} Inhibition of cutinase has been shown to prevent fungal infection through intact cuticles. Conversely, the supplementation of cutinase to fungi that are not able to produce it naturally had been shown to enhance fungal infection success rates.

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.{{cite journal | vauthors = Chen S, Tong X, Woodard RW, Du G, Wu J, Chen J | title = Identification and characterization of bacterial cutinase | journal = The Journal of Biological Chemistry | volume = 283 | issue = 38 | pages = 25854–25862 | date = September 2008 | pmid = 18658138 | doi = 10.1074/jbc.m800848200 | pmc = 3258855 | doi-access = free }}{{Cite journal | vauthors = Fett WF, Wijey C, Moreau RA, Osman SF |date=April 1999 |title=Production of cutinase byThermomonospora fuscaATCC 27730 |journal=Journal of Applied Microbiology |volume=86 |issue=4 |pages=561–568 |doi=10.1046/j.1365-2672.1999.00690.x |s2cid=41512145 |issn=1364-5072}} 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.

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.{{cite journal | vauthors = Martinez C, De Geus P, Lauwereys M, Matthyssens G, Cambillau C | title = Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent | journal = Nature | volume = 356 | issue = 6370 | pages = 615–618 | date = April 1992 | pmid = 1560844 | doi = 10.1038/356615a0 | bibcode = 1992Natur.356..615M | s2cid = 4334360 }} Fungal cutinase is generally composed of around 197 amino acid residues, and its native form consists of a single domain.{{Cite journal | vauthors = Carvalho CM, Aires-Barros MR, Cabral J |date=1998-12-15 |title=Cutinase structure, function and biocatalytic applications |journal=Electronic Journal of Biotechnology |volume=1 |issue=2 |pages=160–173 |doi=10.2225/vol1-issue3-fulltext-8 |issn=0717-3458|url=http://www.bioline.org.br/abstract?id=ej98020 |hdl=1807/2161 |hdl-access=free }} The protein also contains 4 invariant cysteine residues that form 2 disulfide bridges, whose cleavage results in a complete loss of enzymatic activity.{{Cite journal | vauthors = Ettinger WF, Thukral SK, Kolattukudy PE |date=1987-12-01 |title=Structure of cutinase gene, cDNA, and the derived amino acid sequence from phytopathogenic fungi |journal=Biochemistry |volume=26 |issue=24 |pages=7883–7892 |doi=10.1021/bi00398a052 |issn=0006-2960}}

Crystal structures have shown that the active site of cutinases is found on one end of the ellipsoid shape of the enzyme.{{cite journal | vauthors = Jelsch C, Longhi S, Cambillau C | title = Packing forces in nine crystal forms of cutinase | journal = Proteins | volume = 31 | issue = 3 | pages = 320–333 | date = May 1998 | pmid = 9593202 | doi = 10.1002/(sici)1097-0134(19980515)31:3<320::aid-prot8>3.0.co;2-m | s2cid = 29668663 }} This active site is seen flanked by two hydrophobic loop structures and partly covered by 2 thin bridges formed by amino acid side chains. It does not possess a hydrophobic lid, which is a common constituent feature among other lipases. 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. Cutinase activation is believed to be derived from slight shifts in the conformation of hydrophobic residues, acting as a miniature lid. 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.{{cite journal | vauthors = Nicolas A, Egmond M, Verrips CT, de Vlieg J, Longhi S, Cambillau C, Martinez C | title = Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state | journal = Biochemistry | volume = 35 | issue = 2 | pages = 398–410 | date = January 1996 | pmid = 8555209 | doi = 10.1021/bi9515578 }}

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.{{cite journal | vauthors = Martinez C, Nicolas A, van Tilbeurgh H, Egloff MP, Cudrey C, Verger R, Cambillau C | title = Cutinase, a lipolytic enzyme with a preformed oxyanion hole | journal = Biochemistry | volume = 33 | issue = 1 | pages = 83–89 | date = January 1994 | pmid = 8286366 | doi = 10.1021/bi00167a011 }} 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. These hydrophobic residues show a higher degree of flexibility, suggesting an induced fit model to facilitate cutin bonding to the active site. In the cutinase active site, histidine deprotonates serine, allowing the serine to undergo a nucleophilic attack on the cutin carboxylic ester.{{Cite web | vauthors = Leung M, Holliday G, Willey J |title=Cutinase |url=https://www.ebi.ac.uk/thornton-srv/m-csa/entry/631/ |access-date=2022-09-28 |website=Mechanism and Catalytic Site Atlas |language=en}} 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. The process repeats with a nucleophilic attack on the new carboxylic ester by a deprotonated water molecule. Following this, the charged oxygen reforms its double bond, removing the serine attachment and releasing the carboxylic acid R monomer.

File:Cutinase Hydrolysis Mechanism.png

The stability of cutinases in higher temperatures (20-50 °C) and its compatibility with other hydrolytic enzymes has potential applications in the detergent industry.{{Cite journal | vauthors = Dutta K, Sen S, Veeranki VD |date=February 2009 |title=Production, characterization and applications of microbial cutinases |journal=Process Biochemistry |volume=44 |issue=2 |pages=127–134 |doi=10.1016/j.procbio.2008.09.008 |issn=1359-5113}} 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.{{cite book | vauthors = Egmond MR, van Bemmel CJ | title = Impact of structural information on understanding lipolytic function | chapter = [6] Impact of structural information on understanding lipolytic function | series = Methods in Enzymology | volume = 284 | pages = 119–129 | date = 1997 | pmid = 9379930 | doi = 10.1016/s0076-6879(97)84008-6 | publisher = Elsevier | isbn = 9780121821852 }} 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. 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.

Rather unexpectedly, the ability to degrade the cutin layer of plants and their fruits holds the potential to be beneficial to the fruit industry. 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.{{Cite journal |date=January 1997 |title=5510131 Enzyme assisted degradation of surface membranes of harvested fruits and vegetables |journal=Biotechnology Advances |volume=15 |issue=1 |pages=273 |doi=10.1016/s0734-9750(97)88551-5 |issn=0734-9750}} 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.

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• PETase

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• {{cite journal|pmc=3294458 |date=2012 |last1=Sulaiman |first1=S. |last2=Yamato |first2=S. |last3=Kanaya |first3=E. |last4=Kim |first4=J. J. |last5=Koga |first5=Y. |last6=Takano |first6=K. |last7=Kanaya |first7=S. |title=Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach |journal=Applied and Environmental Microbiology |volume=78 |issue=5 |pages=1556–1562 |doi=10.1128/AEM.06725-11 |pmid=22194294 |bibcode=2012ApEnM..78.1556S }}
• {{cite journal|doi=10.1021/acs.biochem.7b01189 |title=Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis |date=2018 |last1=Shirke |first1=Abhijit N. |last2=White |first2=Christine |last3=Englaender |first3=Jacob A. |last4=Zwarycz |first4=Allison |last5=Butterfoss |first5=Glenn L. |last6=Linhardt |first6=Robert J. |last7=Gross |first7=Richard A. |journal=Biochemistry |volume=57 |issue=7 |pages=1190–1200 |pmid=29328676 }}

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{{Esterases}}

{{Enzymes}}

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Category:EC 3.1.1

Category:Enzymes of unknown structure

Category:Protein domains