cutinase
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The enzyme cutinase (
systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivial ...
: cutin hydrolase,''
EC 3.1.1.74
is a member of the hydrolase family. It catalyzes the following reaction: R1COOR2 + H2O -> R1COOH + R2OH In biological systems, the reactant carboxylic
ester In chemistry, an ester is a compound derived from an oxoacid (organic or inorganic) in which at least one hydroxyl group () is replaced by an alkoxy group (), as in the substitution reaction of a carboxylic acid and an alcohol. Glycerides ...
is a constituent of the
cutin Cutin is one of two waxy polymers that are the main components of the plant cuticle, which covers all aerial surfaces of plants. It is an insoluble substance with waterproof quality. Cutin also harbors cuticular waxes, which assist in cuticle st ...
polymer A polymer (; Greek '' poly-'', "many" + '' -mer'', "part") is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...
, and the
hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile. Biological hydrolysi ...
of cutin results in the formation of alcohol and
carboxylic acid In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is or , with R referring to the alkyl, alkenyl, aryl, or other group. Carboxyli ...
monomer In chemistry, a monomer ( ; '' mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization. Classification ...
products.


Nomenclature

Cutinase has an assigned
enzyme commission number The Enzyme Commission number (EC number) is a numbering scheme, numerical classification scheme for enzymes, based on the chemical reactions they catalysis, catalyze. As a system of enzyme nomenclature, every EC number is associated with a recomme ...
of EC 3.1.1.74. Cutinase is in the third class of enzymes, meaning that its primary function is to hydrolyze its substrate (in this case, cutin). 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.


Function

Most plants have a layer composed of cutin, called the cuticle, on their aboveground surfaces such as stems, leaves, and fruits. This layer of cutin is formed by a matrix-like structure that contains waxy components embedded in the carbohydrate layers. The molecule, cutin, which composes most of the cuticle matrix (40-80%), is composed primarily of
fatty acid In chemistry, particularly in biochemistry, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, f ...
chains that are polymerized via carboxylic ester bonds. Research suggests that cutin plays a critical role in preventing
pathogen In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a g ...
ic infections in plant systems. 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
spore In biology, a spore is a unit of sexual or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plants, algae, ...
s.; Cutinase is produced by a variety of fungal plant pathogens, and its activity was first detected in the fungus, ''Penicillium spinulosum''. 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 Positive feedback (exacerbating feedback, self-reinforcing feedback) is a process that occurs in a feedback loop which exacerbates the effects of a small disturbance. That is, the effects of a perturbation on a system include an increase in th ...
loop until the fungus successfully breaches the cutin layer; however, the specific mechanism of this pathway is unclear. 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
bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...
l 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. 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.


Structure

Cutinase belongs to the α-β class of proteins, with a central
β-sheet The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a gen ...
of 5 parallel strands covered by 5
alpha helices The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
on either side of the sheet. Fungal cutinase is generally composed of around 197
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
residues, and its native form consists of a single domain. The protein also contains 4 invariant cysteine residues that form 2 disulfide bridges, whose cleavage results in a complete loss of enzymatic activity.
Crystal structure In crystallography, crystal structure is a description of the ordered arrangement of atoms, ions or molecules in a crystalline material. Ordered structures occur from the intrinsic nature of the constituent particles to form symmetric pattern ...
s have shown that the
active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate ( binding site) ...
of cutinases is found on one end of the ellipsoid shape of the enzyme. This active site is seen flanked by two
hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, ...
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
lipase Lipase ( ) is a family 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 ...
s. Instead, the catalytic
serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − for ...
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 An oxyanion hole is a pocket in the active site of an enzyme that stabilizes transition state negative charge on a deprotonated oxygen or alkoxide. The pocket typically consists of backbone amides or positively charged residues. Stabilising the t ...
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.


Mechanism

Cutinase is a serine esterase, and the active site contains a serine-
histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the ...
-
aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
triad and an oxyanion hole, which are signature elements of serine hydrolases. The binding site of the cutin lipid polymer consists of two hydrophobic loops characterized by nonpolar amino acids such as
leucine 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- ...
,
alanine Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side ...
,
isoleucine Isoleucine (symbol Ile or I) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depr ...
, and
proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the p ...
. These hydrophobic residues show a higher degree of flexibility, suggesting an
induced fit Enzyme catalysis is the increase in the rate of a process by a biological molecule, an "enzyme". Most enzymes are proteins, and most such processes are chemical reactions. Within the enzyme, generally catalysis occurs at a localized site, call ...
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. 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.


Applications

The stability of cutinases in higher temperatures (20-50 °C) and its compatibility with other hydrolytic enzymes has potential applications in the
detergent A detergent is a surfactant or a mixture of surfactants with cleansing properties when in dilute solutions. There are a large variety of detergents, a common family being the alkylbenzene sulfonates, which are soap-like compounds that are m ...
industry. 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. 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 Biofuel is a fuel that is produced over a short time span from biomass, rather than by the very slow natural processes involved in the formation of fossil fuels, such as oil. According to the United States Energy Information Administration ...
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. 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.


References


See also

*
PETase PETases are an esterase class of enzymes that catalyze the hydrolysis of polyethylene terephthalate (PET) plastic to monomeric mono-2-hydroxyethyl terephthalate (MHET). The idealized chemical reaction is (where n is the number of monomers i ...
{{InterPro content, IPR000675 EC 3.1.1 Enzymes of unknown structure Protein domains