Which Enzymes Catalyse the Deacetylation of Drugs in the Human Body?

Which Enzymes Catalyse the Deacetylation of Drugs in the Human Body?

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If you would like more specifics seeing how I realise that this question is very broad and may be difficult to answer in general then hopefully the following will help you out:

  1. I am particularly interested in acetyl groups bound by carbon single bonds
  2. Drug metabolism in the liver particularly interests me
  3. The drug paracetamol's (acetaminophen) deacetylation to p-aminophenol is of particular interest to me.

Acetyl esters can be deacetylated by carboxylesterases.

In case of an acyl/aryl group attached to acetate group (e.g., benzoate), I guess it will first undergo ring opening and then beta oxidation (as in case of tyrosine).

Paracetamol -> p-aminophenol is a deamidation (I am not sure, but enzymes like ornithine decarboxylase can do that job… just a guess).

Biopolymers as biofilters and biobarriers Effect of degree of deacetylation of biopolymer

Deacetylation of biopolymer escalates the NOB immobilization on the biopolymer surface. Figure 17.6 exhibits that NOB attached on chitosan with either degree of deacetylation (DD) 82% or DD 91% (molecular weight of 310,000–410,000 g/mol) showed a considerably higher nitrite reduction rate compared to that of chitin. For instance, NOB attached on chitosan with a DD of 91% can reduce nitrite level at a rate of 0.82 ± 0.05 mg-N/(g·day), while NOB attached on chitin simply accomplished 0.44 ± 0.03 mg-N/(g·day). Chitosan with DD of 82% and 91% does not have a substantial effect on nitrite reduction rate (P > 0.05). In other words, chitosan employed as a biofilter should have DD exceeding 80%. This fact can be clarified by charge density. After pretreatment of the surface of chitosan with a buffer at pH 6.5, chitosan having DD exceeding 80% comprises higher cationic charges than chitin, which has a DD of 10–15% ( Lertsutthiwong et al., 2002 ). This leads to strong ionic bonding with the anionic surface of the bacterial cell wall ( Lertsutthiwong et al., 2013 ).

Figure 17.6 . Effect of the degree of deacetylation of chitosan on the rate of nitrite reduction ( Lertsutthiwong et al., 2013 ).

Enzymes In The Body

Enzymes are specific organic molecules found in biological systems that allow cellular life to exist and function at earth temperatures. Most life-supporting chemical reactions could only occur above 90°C or 200°F in the absence of enzymes.[1] Enzymes are referred to as macromolecular biological catalysts. They allow the existence of reactions that would not occur otherwise under numerous conditions having to do with temperature, pH and atmospheric conditions within the human body. Metabolic processes within cells require enzyme catalysts in order to occur at rates fast enough to support life. Enzymes are known to catalyze more than 5,000 biochemical reaction types.[2] The study of this complex topic is called enzymology.

Enzymes accelerate the chemical reaction rate in numerous ways, lowering activation energy. Activation energy is the energy which must be available to a chemical or nuclear system with potential reactants to produce a reaction or product. Enzymes react with other substances, either to take them apart or join them together.[3] They do not alter the position of the chemical equilibrium of the reaction.
During the presence of an enzyme, the reaction moves in the same direction as it would without the enzyme, however enzyme presence accelerates the process.

Function and Nature of Enzymes
Enzymes are responsible for:
1. Signal transduction and cell regulation often by kinases and phosphatases.
2. Generating movement with myosin (muscle protein) hydrolyzing ATP to generate muscle contraction.
3. Transporting cargo around the cell as part of the cytoskeleton.
4. Digestion, metabolism, respiration
5. Digestive enzymes such as amylases and protease break down large molecules of starches or proteins into smaller ones for proper absorption in the intestines.
6. Hormone production.
7. Nutrient absorption and transportation.
8. Cellular repair and division.
9. Detoxification
10. Disease: Viruses can contain enzymes for infecting cells, i.e. HIV integrase and reverse transcriptase.

1. Stabilize the transition state: a. Create an environment with a charge distribution complementary to that of the transition state to lower its energy.
2. Provide an alternative reaction pathway: a. Temporarily reacts with the substrate, forming a covalent intermediate to provide a lower energy transition state.
3. Destabilizes the substrate ground state: a. Distort bound subtract(s) into their transition state form to reduce the energy required to reach the transition state. b. Orient the substrates into a productive arrangement to reduce the reaction entropy (thermodynamic) change.
Enzymes essentially react with other substances, either to take them apart or join them together.
Enzymes are divided as [4] :
1. Simple: contains the protein part only (e.g., hydrolases like pepsin, trypsin or ribonuclease).
2. Complex: Proteins may be joined with a non-protein part, referred to as prosthetic groups. The protein part is called the apoenzyme. The non-protein part is referred to as a Cofactor. Together, apoenzyme and Cofactor, form a biologically active molecule of enzyme – the holoenzyme.
a. Metal ion: helps the enzyme to position the substrate molecule into the active site. Called activators, the associated metals may include copper, cobalt, zinc, magnesium, molybdenum and manganese.
b. Organic molecule: often vitamins such as riboflavin, the B vitamins and vitamin C.
c. Coenzymes: a non-protein organic molecule that binds to the molecule of apoenzyme freely, thus can detach from it, i.e. NAD+ (Nicotinamide adenine dinucleotide) and NADP+ (Nicotinamide adenine dinucleotide phosphate). NAD+ and NADP+ are electron carriers in cellular respiration. NADP+ is created in anabolic reaction, or reaction that build large molecules from small molecules.
d. Prosthetic group: a non-protein organic molecule that binds to the molecule of apoenzyme tightly, i.e. heme, FAD (flavin adenine dinucleotide)

A substrate
The molecules upon which enzymes react is called a substrate. The enzyme remains intact and is not consumed during chemical reactions. Nor do they alter the stability of a reaction. Instead, they support the progression of a reaction maintaining equilibrium. The majority of enzymes are proteins made up of amino acids, the basic building blocks within the body.
There are exceptions with some kinds of RNA molecules called ribozymes.[5] Amino acid molecules are connected through linkages known as peptide bonds that form proteins.
Enzymes are made up of a different number of peptide chains and are termed multienzyme complexes. An example of multienzyme complexes would be the fatty acid synthase, an enzyme catalyzing the synthesis of higher fatty acids in cells.[6] Chemically, the small groups of bonded amino acids are called polymeric molecules or referred in the biochemistry as poly peptides.
It takes 30 amino acids to form a long enough chain that enable the molecules to influence its own shape in becoming a protein. Whether one chain or multiple chains, it contains multiple parts called domains. Proteins serve numerous biochemical functions including anatomical structural features in organisms, nutrient carriers, antigens and hormones.[7]

The Enzyme – Substrate Complex
Coined as a “Lock and key” model, enzymes react with other substances to either take them apart or join them together. [8]There is a high specificity of enzymes demonstrated by the way they associate or bind with the substrate: the reactant molecule. Chemoselective, every enzyme molecule has an active site on its surface. The reactant molecule is attracted to and molded into the indentation that forms the active site.
Specificity is achieved by binding pockets with complementary, shape, charge and hydrophilic/hydrophobic characteristic to the substrates. The active site continues to change until the substrate is completely bound, resulting in a final shape.
That said, enzymes are flexible structures and the active site is continuously reshaped by interaction with the substrate as it interacts with the enzyme.

Controlling Enzyme Activity
Enzymes guide and regulate metabolism of a cell and are carefully controlled.[9] The mechanism of action of enzymes involve regulatory molecules that can either increase (activator) or inhibit (inhibitors) the activity of an enzyme. An enzyme inhibitor is a molecule that binds to an enzyme and blocks the binding of a substrate, decreasing its activity. If an enzyme produces too much of a substance in an organism, that substance begins to act as an inhibitor for the enzyme at the beginning of the pathway as a form of negative feedback, slowing the reaction down. Drugs can be enzyme inhibitors. For example, blocking an enzyme’s activity can kill a pathogen or correct a metabolic imbalance.[10]

The control of enzyme activity is essential for homeostasis in the body. When there is malfunction of an enzyme such as a mutation, over or under production, or deletion, this can lead to a genetic disease. In some cases, it can be fatal. For example, pancreatic insufficiency is a condition which occurs when the pancreas does not make enough of a specific enzyme required to digest food in the small intestine.[11]

Table 1 - Factors affecting or control enzyme activity [12]

Temperature modulation
The catalytic activity of enzymes requires optimum temperature within the body. Human enzymes have maximal activity at 37oC. Enzymes can become vulnerable to temperature changes. Due to their protein nature, applying high temperature between 55-60o C causes denaturing of protein, producing a conformational change and destruction of protein. This change causes a drop in or a complete halt of the reaction.[13] Moreover, low temperatures can slow reactions, reducing the activity of enzymes.

Enzymes are sensitive to the change in pH. As in temperature changes, extremes of low and high again lead to denaturation of the molecules. Concentration of H+ affects the ionization of acidic and base groups

Enzyme Sources [14]
1. Metabolic enzymes: regulates organs, tissue, and blood. Help create new cells, repair existing damaged cells and moves nutrients to where you body most needs them.
2. Digestive Enzymes: breaks down food. Subtypes – amylase, lipase, protease
3. Raw foods: Supports the immune system, cellular repair.
There are six major classes of enzymes with specific function. There are also subgroups.

Table 2 – Enzyme types [15,16]

Enzyme subgroups:
Hydrolases are enzymes that split out water, separating the parts of molecules.
Hydrogenases are enzymes that add hydrogen atoms to other molecules 5 alpha reductase is an example.
Oxidases catalyze oxidations by adding oxygen or electrons to molecules or atoms.

Enzymes in the Skin Building of the skin barrier and the final desquamation process
The entire epidermal differentiation process is dependent upon enzymatic activity. Lipid hydrolases are responsible for conversion of lipids into ceramides and free fatty acids. Enzymes are involved in profilaggrin modification and proteolytic processing in the epidermis. [17] The healthy transitional phases of the entire building process of the stratum corneum is vital and dependent upon enzymes.

Protease enzymes are essential to the normal desquamation process within the cells of the stratum corneum (SC). Desmosomes are important for strong cell-to-cell adhesion. The desquamation process involves the proteolysis of intercellular adhesive structures created in the desmosome structures. There are specific protease enzymes within the stratum corneum called tryptic enzyme and chymotryptic enzyme involved with the degradation of the corneodesmosomes. Two proteins found in desmosomes are desmoglein and desmocollin, located at the interconnections within cells.
The stratum corneum chymotryptic enzyme is produced as an inactive precursor with no proteolytic activity. Hence there is a requirement of an activating enzyme that involves an enzyme with trypsin-like substrate. Different proteases attack the different amino acid sites on the desmoglein and desmocollin proteins. When bonds are weakened, they break, allowing cells to flake off.
The desquamation process requires water from within the epidermis and a normal pH. The orchestration of all biological activities is complex and must work synergistically with one another.

Structure, mechanism, and inhibition of histone deacetylases and related metalloenzymes

Metal-dependent histone deacetylases (HDACs) catalyze the hydrolysis of acetyl-L-lysine side chains in histone and nonhistone proteins to yield l-lysine and acetate. This chemistry plays a critical role in the regulation of numerous biological processes. Aberrant HDAC activity is implicated in various diseases, and HDACs are validated targets for drug design. Two HDAC inhibitors are currently approved for cancer chemotherapy, and other inhibitors are in clinical trials. To date, X-ray crystal structures are available for four human HDACs (2, 4, 7, and 8) and three HDAC-related deacetylases from bacteria (histone deacetylase-like protein (HDLP) histone deacetylase-like amidohydrolase (HDAH) acetylpolyamine amidohydrolase (APAH)). Structural comparisons among these enzymes reveal a conserved constellation of active site residues, suggesting a common mechanism for the metal-dependent hydrolysis of acetylated substrates. Structural analyses of HDACs and HDAC-related deacetylases guide the design of tight-binding inhibitors, and future prospects for developing isozyme-specific inhibitors are quite promising.

Copyright © 2011 Elsevier Ltd. All rights reserved.


Figure 1. Arginase-deacetylase fold

Figure 1. Arginase-deacetylase fold

(a) Topology diagrams of arginase, HDAC8, and APAH reveal a common…

100D (boldface indicates metal ligands). The Mn 2+ A site of arginase is not conserved in HDACs or HDAC-related deacetylases. Non-protein metal ligands (red spheres) are solvent molecules in arginase and HDAC8, and the oxygen atoms of a hydroxamate inhibitor in APAH.

Medicinal Chemistry Approaches to Malaria and Other Tropical Diseases

Sandra Gemma , . Giuseppe Campiani , in Annual Reports in Medicinal Chemistry , 2019

4.5.2 Epigenetic targets

Histone modifying enzymes are involved in the post-translational modification of histone and non-histone substrates and are involved in the epigenetic control of important cellular functions. 71 In particular, histone deacetylases (HDACs) are involved in control of gene expression, cell proliferation, angiogenesis, etc. Human HDAC enzymes have been classified into different classes: Class I comprising HDAC1, -2, -3, and -8, class IIa comprising HDAC4, -5, -7, and -9, class IIb comprising HDAC6 and -10, and class IV comprising HDAC11. Class III enzymes are called sirtuins and use nicotinamide adenine dinucleotide as co-factor, while class I, II and IV are zinc-dependent enzymes. 72 HDAC inhibitors are approved for the treatment of cancer. However, various HDAC inhibitors have demonstrated antiparasitic activity in malaria, leishmania, trypanosomiasis and schistosomiasis, clearly highlighting the key role of HDAC orthologues in eukaryotic parasites. 73,74 Pan-HDAC inhibitors have been tested against schistosome and have been proven to be active in killing both schistosomula and adult worms. 75,76 Moreover, the downstream effect of histone deacetylation has been in depth studied. 77,78 In S. mansoni three class I (SmHDAC1, -3, and -8) and 5 class III (SmSirt1, -2, -5, -6, and -7) HDACs have been characterized. 79

Sirtuins catalyze the deacetylation of acetylated ɛ-aminogroups of lysines in histones and other proteins. The search for sirtuin inhibitors specifically designed against Schistosoma has been hampered by the lack of suitable screening assays. Schiedel 80 recently described a novel assay for SmSirt2 activity that should lead to the implementation of screening campaign against this target.

More advanced is the status of other HDAC classes such as SmHDAC8, as targets for the development of anti-schistosoma therapies. 81 Starting from the X-ray co-crystal structure of SmHDAC8 in complex with inhibitor 59 ( Fig. 12 ), 82 rational design approach resulted in the synthesis of a first series of inhibitors characterized by a 3-amino- and 3-amidobenzohydroxamic acid scaffold typified by 60 and 61. The X-ray crystal structure of 61/SmHDAC8 was again solved and used for further derivatization and rational design. Beside the expected interaction of the hydroxamate group with Zn ion, a clamp formed by SmHDAC8 Lys20 and His292 with the amido group of 61 looked highly specific for the interaction of this compound with SmHDAC8. Subsequent SAR and docking studies highlighted that halogens or methoxy substituents at the four position increased activity and also bulkier aromatic rings such as biphenyl and quinolinamides. Docking studies confirmed the key role played by H292 forming an H-bond with the amido group and that is replaced by a methionine in the human orthologue. 83 Optimized inhibitor 62 (smHDAC8 IC50 = 75.4 nM, hHDAC8 IC50 = 26.1 nM, hHDAC1 IC50 = 6.3 μM, and hHDAC6 IC50 = 390 nM) was then submitted to phenotypic assays showing that this compound is lethal to schistosomula after 2 days of incubation at 10 μM. On adult worms, 62 caused marked separation of adult male and female worm pairs and a decrease in egg production at 20 μM. However, low selectivity against human orthologues represents an issue for this class of compounds. Starting from these studies and guided by X-ray crystallography and docking, further elaboration of the scaffold resulted in the identification of isophthalic acid derivatives typified by 63 84 (smHDAC8 IC50 = 330 nM, hHDAC8 IC50 = 90 nM, hHDAC1 IC50 = 136.1 μM, and hHDAC6 IC50 = 2.9 μM) and cinnamic acid derivatives such as 64 85 (smHDAC8 IC50 = 90 nM, hHDAC8 IC50 = 80 nM, hHDAC1 IC50 = 11.5 μM, and hHDAC6 IC50 = 6.1 μM) as compounds with good activity against SmHDAC8 and acceptable selectivity profile against the human orthologues HDAC1 and HDAC6. Virtual screening based on structure-based 3D-QSAR model or docking-based procedures identified novel structural compounds but endowed with high micromolar activity against SmHDAC8 and low selectivity over human orthologues, highlighting the need of further optimization of the discovered hit compounds. 86,87

Fig. 12 . SmHDAC8 inhibitors.

Mammalian deacetylases

Among mammalian class I deacetylases (subtypes 1, 2, 3 and 8), HDACs 1 and 2 are most closely related (82% sequence identity) and found in the ubiquitously expressed mSin3A, NURD/Mi2/NRD and CoREST corepressor complexes 28 . HDAC1 KO mice die during embryonic development and their ES cells show decreased growth and enhanced expression of the cyclin-dependent kinase inhibitors p21 and p27 29 . Compensatory upregulation of the expression of other class I HDACs in HDAC1 KO cells is apparently insufficient to counterbalance the loss of this subtype, suggesting both its functional uniqueness and the existence of regulatory cross-talk 30 . Both HDACs 1 and 2 (in addition to HDAC3) seem to be involved in the regulation of key cell cycle genes such as p21 ( 31, 32, 33 and MP et al., manuscript in preparation). Still, HDAC2 also has some specialized functions, e.g. in counterbalancing proapoptotic signals that ensue from aberrant Wnt pathway activation in colon cancer 34 . HDACs 1 and 2 are not exclusively HDACs but may deacetylate non-histone substrates as well 35 .

HDAC3 associates to and is activated by SMRT and NCoR co-repressors that play an important role in the regulation of gene expression by nuclear hormone receptors 28 , which, in their unliganded state, recruit these corepressors and utilize the deacetylase activity to silence transcription. Studies on the retinoic acid receptor suggest that a specific lysine (H4K5) is preferentially deacetylated by HDAC3 recruited to the unliganded receptor 36 . Intriguingly, the SMRT/N-CoR corepressors are themselves capable of interacting with histones via an SANT domain and this interaction is strengthened upon deacetylation of H4K5, suggesting the existence of a positive feedback loop in the silencing mechanism. HDAC3 was found to be upregulated in CD34 + CD133 + early hematopoietic progenitors 37 and to play a transcription-independent role in mitosis 38 , possibly pointing to functions in cell cycle progression and stem cell self-renewal. In contrast to HDACs 1 and 2 that are exclusively nuclear, HDAC3 may also be found in the cytoplasm and even associated with the plasma membrane, where it can be phosphorylated by Src 39 . Also HDAC3 is not an exclusive HDAC but may also deacetylate non-histone substrates, such as the RelA subunit of NF-κB, thereby affecting its stability and DNA-binding properties 40, 41 . The last member of class I, HDAC8, was recently found to be expressed in smooth muscle where it is required for muscle contractility 42 . This protein has also been linked to cancer since it is recruited by the leukemic INV 16 protein 43 it regulates telomerase activity 44 and siRNAs targeting HDAC8 were shown to have antitumor effects in cell culture 45 .

The class IIa HDACs (subtypes 4, 5, 7 and 9) are characterized by tissue-specific expression and stimulus-dependent nucleo-cytoplasmic shuttling 19 . They are target of several kinases, and some phosphorylated forms are confined to the cytosol by interaction with 14-3-3 proteins (see below). In the nucleus they associate with transcription factors, notably of the MEF and Runx families, and control differentiation and cellular hypertrophy in muscle and cartilage tissues 46, 47 . HDAC4 KO mice have a pronounced chondrocyte hypertrophy and die of aberrant ossification 46 . HDAC9 KO mice show cardiac hypertrophy 47 that is further exacerbated in the HDAC9+HDAC5 double KO animals 48 . HDAC7 has a specific role in the clonal expansion of T cells by suppressing Nur77-dependent apoptosis 49 and in vascular integrity through suppression of MMP10 50 . Class IIb subtypes 6 and 10 have a duplication of their catalytic domains, but the second catalytic domain is thought to be dysfunctional in HDAC10. HDAC6 is the only deacetylase known to act on tubulin 51, 52 . Tubulin deacetylation is required for disposal of misfolded proteins in aggresomes 53 . HDAC6 also deacetylates Hsp90, pointing to a broader role of this subtype in protein folding 54, 55, 56 . Finally, very little is known about HDAC11, which cannot be clearly assigned to either class I or class II HDACs based on sequence motifs 57 .

Enzymes in the Body and their Functions

There are three types of enzymes food enzymes, digestive enzymes and metabolic enzymes. These enzymes are explained in the following paragraphs:

Food Enzymes

Food enzymes are present in all raw foods like animal or plant products. The names of enzymes that are plant-based are protease, lipase, amylase and cellulase. They contain active units that help break down fat, proteins and carbohydrates in the body at the broadest range of pH within the body. They also help in maintaining a proper digestive system and help the body produce more metabolic enzymes. Pepsin, bromelain, etc. are animal based enzymes that help in digestion, as an anti-inflammatory agent. Trypsin helps in braking down arginine or lysine and is active at alkaline pH. The other enzymes that carry out chemical reactions are rennin that readies the milk for the action of pepsin and lipase by braking it down to proteins and fats.

Digestive Enzymes

Digestive enzymes are secreted by the body that helps in digestion of food. The names of enzymes that help in digestion are:

  • Amylase: This enzyme helps in breaking down carbohydrates. It is found in saliva, pancreas and intestinal juices.
  • Proteases: It helps in digestion of proteins. It is present in the stomach, pancreatic and intestinal juices.
  • Lipases: Lipases assist in digestion of fats. It is seen in the stomach, pancreatic juice and food fats.

Amylase I and II are secreted by the salivary glands initially and then by the pancreas. They break the bonds between carbohydrate molecules and produce disaccharides and trisaccharides. Amylase I is activated by chewing and converts starch to maltose. Amylase II is secreted only by the pancreas and carries on with the process that has been initiated with Amylase I.

Pepsin is produced as a proenzyme pepsinogen by the chief cells of the stomach. It gets activated by the hydrogen in the stomach and produces hydrochloric acid at the same time. It breaks the bonds between amino acids in the proteins and produces short chain polypeptides. It also kills any pathogen that enters the body through food.

Pancreas produce trypsin as a proenzyme trypsinogen. It works on polypeptides and proteins producing short chain peptides. It is also acts as an activating enzyme for other pancreatic proteinases. Chymotrypsin produced by the pancreas acts on proteins and polypetides producing short-chain peptides.

Pancreas produce carboxypeptidase as proenzyme procarboxypeptidase. It acts on proteins and polypeptides producing short-chain peptides and amino acids. Another enzyme produced by the pancreas is elastase, that acts on elastin producing short chain of peptides. If there are bile salts present, the pancreas produce lipase that targets triglycetides producing fatty acids and monoglycerides. Vitamin C, glutathione and cysteine play important roles in activation of lipase.

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Nuclease produced by pancreas acts on nucleic acids like RNA and DNA to produce nitrogen bases and simple sugars. The mucosal cells of the small intestine secrete enterokinase that reaches the lumen by shedding of epithelial cells. It acts on trypsinogen to produce trypsin. Mucosal cells of small intestines also produce maltase, sucrase and lactase to target sugars like maltose, sucrose and lactose to produce monosaccharides. Peptidase is another enzyme in the body produced by mucosal cells of small intestine that target dipeptides and tripeptides producing amino acids.

Metabolic Enzymes

The metabolic enzymes are found moving all over the body systems and organs. They carry out many chemical reactions within the body cells. Superooxide dismutase, an antioxidant and catalase, the enzyme that breaks down hydrogen peroxide are two most important metabolic enzymes.

These are just a few of the many enzymes in the body and their functions. Enzymes are necessary for cellular functions, completion of digestion, nutrient absorption, combating free radicals and supporting liver detoxification. There are many enzymes that are not produced by our body and need to be supplemented through external sources. Thus, it is essential to maintain a healthy diet . There are many enzyme supplements available in the market that can help you overcome deficiencies, under medical supervision. Excessive intake of enzymes may lead to headaches, bloating, acne, gas, etc.

There are innumerable functions of enzymes, other than those mentioned in this article. Our blood is prevented from getting clot in certain parts of the body by a fibrinolytic enzyme. There are many such chemical reactions that help in the normal functioning of the body. Thus, enzymes in the body can be called the hidden heroes of a well-functioning body, without whom the body will cease to operate.

Bio-Technological Applications of Enzymes

Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes.

Biotechnology could be traced back to thousands of years ago when human started to use yeasts to make liquor. This may be the first dawn of biotechnology in food production. Along with the development of natural and social sciences, nowadays, biotechnology carries more colourful meanings.

In the modern world, biotechnology often refers to the process of making or modifying products using living systems or organisms. Besides the traditional fermentation, biotechnological tools have expanded to employ more advanced sciences, such as genetic engineering, applied immunology, and medicinal therapies and diagnostic. Among these tools, a variety of enzymes are indispensable—they may appear as hammers and chisels to help accomplish molecular biology experiments, or they may act as efficient micro-reactors in industrial production. They exist everywhere and resemble all kinds of functions in living cells or in harsh environments bearing no life. As of today, people exploit their functions in great details for better uses in biotechnology.

Enzymes in the applications of biotechnology

A majority of drinks and foods in our daily lives are dependent on the food production industry. The transformation of milk to cheese, grains to alcohol, fruits to juice, and flour to bread all rely on the reactions catalysed by enzymes. Because of the “green chemistry” characterised by enzymatic catalysis, these reactions can be used to produce food grade products while avoiding residues of harmful substances. As a result, using enzymatic biotechnology in food production has been rapidly developed. In addition to the superior catalytic activity, high selectivity of enzymes is another deeply admired merit. For example, maltose is a disaccharide composed of two units of glucose, which has many structurally similar isomers. Traditionally, it was almost impossible to separate maltose from its isomers, and a mixture of these isomers was used as one raw material, making quality control difficult. Now, enzymes can specifically transform each isomer to different molecules, meeting different needs in flavoring, nutrition, chemistry, and pharmaceuticals. Especially, trehalose synthase only reacts with maltose and converts it to trehalose, which is used as a food additive and biological agent.

The rapid development of technologies in gene engineering allows modifications of biological activities at molecular levels. Biotechnology in medical research and agriculture becomes a busy area. Enzymes have various roles in medical research, including new drug development, disease treatment, and diagnosis or prognosis. Enzymes can also be applied to assay kits, such as coupled multi-enzyme assay kits and enzyme-linked immunosorbent assay (ELISA) kits, to conveniently conduct tests and diagnosis.

Lots of pharmaceuticals share chiral preferences therefore, the synthesis and development of chiral molecules are the vital parts of pharmaceutical manufacture. In recent years, using enzymes becomes popular in achieving chiral resolution, instead of chemical catalysis. The enzymatic route wins this application due to high selectivity and specificity of enzymatic reactions. Optical isomers containing both R- and S- configurations are often present as the final products of chemical catalysed reactions. As a comparison, enzymes can easily yield chiral and pure molecules, and thus, solve the problem that bothers the traditional chemical synthesis for decades. Hitherto, many successful stories have already been demonstrated. For example, the pro-drug compactin is catalysed by cytochrome oxidase to form pravastatin, which is a treatment of cholesterol-related diseases.

As enzymes are specific biological catalysts, they should make the most desirable therapeutic agents for the treatment of metabolic diseases. Although adverse immune responses may be caused by enzymes from sources foreign to the human body, the advanced techniques of genetic engineering can modify enzyme molecule to reduce or even avoid the antigenicity. A majority of enzymes are used as therapeutic agents. For example, asparaginase and glutaminase are used to treat leukaemia, collagenases are used to treat skin ulcers, and hyaluronidases are used in a heart attack therapy. In the future, there will be more potential enzyme drugs for the treatment of cancer and neurological pathology, which are considered as the next biggest health challenges in the world.

“Diagnostic enzymes” refers to enzymes used for diagnosis or prognosis. How are enzymes used in diagnosis? Because their high specificity and sensitivity to the substrate, even in the presence of other proteins, many enzymes are already used in the diagnosis of bone diseases, cancer, and liver diseases, examples including alkaline phosphatase (ALP), acid phosphatase (ACP), and alanine transaminase (ALT). Enzymes can also be used in analysis and detection processes, such as coupled multi-enzyme reaction assays and enzyme-linked immunosorbent assays (ELISA). Due to the pivotal role in medical diagnostics, more research will be focused on these enzymes to reach clinical applications.

Chemical reactions often take place under harsh conditions or accompanied by intense heating and massive waste production. However, enzymatic catalysis is far milder and more efficient. Therefore, using enzymatic biotechnology in the chemical industry becomes the more recent trend. In recent years, efforts on finding new enzymes with chiral resolution capability have aroused great interests. Traditional chemical reactions often give racemic mixture products, but in most cases, only one chiral configuration is valuable, while ones are considered as impurities or even hazardous wastes. A well-known disastrous example is “Thalidomide”, which was used as a mixture of two chiral isomers. Unfortunately, one isomer has a severe adverse effect and resulted in phocomelia of the new-born, as later found by scientists. Enzymes can easily achieve chiral resolution thus, solve the problem that is greatly challenging to the traditional chemical method.

Nowadays, environment issues grow worse and become a global concern. Scientists hope to reduce pollutions caused by petroleum and its byproduct in a clean and sustainable way. It has been found that some microorganisms have the right enzymes to degrade petroleum, break down the hydrocarbon chain to smaller fragments, which would help with cleaning oil spills and recycling waste oils. Furthermore, recent research revealed that some enzymes efficiently convert natural products such as soybean and corn oils into biofuels, which would also reduce carbon emission in the future. Enzymes can also alleviate soil and water pollution without generating new wastes. Therefore, enzymes carry researchers’ hope to keep the balance between environment and development.

Enzymes are powerful tools that help sustain a clean environment in several ways. They are utilised for environmental purposes in a number of industries including agro-food, oil, animal feed, detergent, pulp and paper, textile, leather, petroleum, and specialty chemical and biochemical industry. Enzymes also help to maintain an unpolluted environment through their use in waste management. Recombinant DNA technology, protein engineering, and rational enzyme design are the emerging areas of research pertaining to environmental applications of enzymes. The future will also see the employment of various technologies including gene shuffling, high throughput screening, and nanotechnology

Artificial enzymes perform reactions on living cells

Nature has evolved thousands of enzymes to facilitate the many chemical reactions that take place inside organisms to sustain life. Now, researchers have designed artificial enzymes that sit on the surfaces of living cells and drive reactions that could someday target drug therapies to specific organs. They report their results in the Journal of the American Chemical Society.

Metalloenzymes are a class of enzymes that contain a metal ion, such as zinc, iron or copper. The metal ion helps the enzyme speed up, or "catalyze," chemical reactions that would otherwise occur very slowly or not at all. Scientists would ultimately like to develop a method to produce therapeutic drugs only at the sites of specific cells or organs of the human body, which could reduce side effects, and enzymes could help them reach that goal. Wadih Ghattas, Jean-Pierre Mahy and their colleagues set their sights on engineering an artificial enzyme that could catalyze a useful reaction, called the Diels-Alder reaction, right on the surfaces of living cells. Chemists use this reaction to synthesize drugs, agrochemicals and many other molecules.

To make their artificial enzyme, the researchers began with a protein called the A2A adenosine receptor, which is naturally present on the surfaces of some cells in the body. They modified a molecule that binds to this receptor with a copper-containing chemical group that catalyzes the Diels-Alder reaction. When the researchers placed the resulting compound in a culture dish containing living human cells, it attached to the A2A adenosine receptors on the cells, forming an artificial enzyme. This enzyme catalyzed the Diels-Alder reaction with an up to 50 percent yield. The researchers say that in the future, artificial enzymes might be designed that bind to proteins found only on specific cell types, for example, cancer cells. Then, the enzyme could convert an inactive compound into a drug to selectively kill those cells.

The authors acknowledge funding from the French National Research Agency.


Synthetic and natural food antioxidants are used routinely in foods and medicine especially those containing oils and fats to protect the food against oxidation. There are a number of synthetic phenolic antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) being prominent examples. These compounds have been widely uses as antioxidants in food industry, cosmetics, and therapeutic industry. However, some physical properties of BHT and BHA such as their high volatility and instability at elevated temperature, strict legislation on the use of synthetic food additives, carcinogenic nature of some synthetic antioxidants, and consumer preferences have shifted the attention of manufacturers from synthetic to natural antioxidants.[74] In view of increasing risk factors of human to various deadly diseases, there has been a global trend toward the use of natural substance present in medicinal plants and dietary plats as therapeutic antioxidants. It has been reported that there is an inverse relationship between the dietary intake of antioxidant-rich food and medicinal plants and incidence of human diseases. The use of natural antioxidants in food, cosmetic, and therapeutic industry would be promising alternative for synthetic antioxidants in respect of low cost, highly compatible with dietary intake and no harmful effects inside the human body. Many antioxidant compounds, naturally occurring in plant sources have been identified as free radical or active oxygen scavengers.[75] Attempts have been made to study the antioxidant potential of a wide variety of vegetables like potato, spinach, tomatoes, and legumes.[76] There are several reports showing antioxidant potential of fruits.[77] Strong antioxidants activities have been found in berries, cherries, citrus, prunes, and olives. Green and black teas have been extensively studied in the recent past for antioxidant properties since they contain up to 30% of the dry weight as phenolic compounds.[78]

Apart from the dietary sources, Indian medicinal plants also provide antioxidants and these include (with common/ayurvedic names in brackets) Acacia catechu (kair), Aegle marmelos (Bengal quince, Bel), Allium cepa (Onion), A. sativum (Garlic, Lahasuna), Aleo vera (Indain aloe, Ghritkumari), Amomum subulatum (Greater cardamom, Bari elachi), Andrographis paniculata (Kiryat), Asparagus recemosus (Shatavari), Azadirachta indica (Neem, Nimba), Bacopa monniera (Brahmi), Butea monosperma (Palas, Dhak), Camellia sinensis (Green tea), Cinnamomum verum (Cinnamon), Cinnamomum tamala (Tejpat), Curcma longa (Turmeric, Haridra), Emblica officinalis (Inhian gooseberry, Amlaki), Glycyrrhiza glapra (Yashtimudhu), Hemidesmus indicus (Indian Sarasparilla, Anantamul), Indigofera tinctoria, Mangifera indica (Mango, Amra), Momordica charantia (Bitter gourd), Murraya koenigii (Curry leaf), Nigella sativa (Black cumin), Ocimum sanctum (Holy basil, Tusil), Onosma echioides (Ratanjyot), Picrorrhiza kurroa (Katuka), Piper beetle, Plumbago zeylancia (Chitrak), Sesamum indicum, Sida cordifolia,Spirulina fusiformis (Alga), Swertia decursata, Syzigium cumini (Jamun), Terminalia ariuna (Arjun), Terminalia bellarica (Beheda), Tinospora cordifolia (Heart leaved moonseed, Guduchi), Trigonella foenum-graecium (Fenugreek), Withania somifera (Winter cherry, Ashwangandha), and Zingiber officinalis (Ginger).[79]


The exchange reaction we describe demonstrates that the SIR2-like proteins form an enzyme-ADP-ribose intermediate, in agreement with a recent report (10). Our data show that this reaction occurs only in the presence of proteins containing acetyllysine. The enzymes also deacetylate the lysines on these proteins, and this deacetylation is, in turn, dependent on NAD. The exact mechanism by which these reactions occur is not clear. The enzymes must have at least two recognition sites, one for NAD and one for acetyllysine, and two activities, auto-ADP-ribosylation and deacetylation. The crystal structure of these enzymes should prove enlightening because previously characterized deacetylases do not exhibit the dependence on NAD described here. A major unanswered question is whether NAD turns over during the deacetylation. In other words, is an ADP-ribose released for every acetyl group removed, or is a stably ADP-ribosylated enzyme the active deacetylase? Future studies defining enzyme and cofactor turnover numbers should resolve this question.

We see no evidence for transfer of ADP-ribose from the enzymes to proteins such as albumin or histones, contrary to recent reports (9, 10). Although we cannot exclude the possibility that a small amount of ADP-ribosylation might occur, three experiments argue against significant transfer. First, we see no [ 32 P]NAD transfer to recipient proteins by either CobB or HST2. The groups that reported such transfer used very high concentrations of enzyme to observe it. We see significant deacetylation at a 100-fold lower enzyme concentration. This result suggests that deacetylation is the physiologically significant reaction. Second, the H4 visualized in the Triton–acid–urea gel in Fig. 3B shows no sign of ADP-ribosylation after HST2 treatment. If that had occurred, the H4 isoforms would be expected to migrate more slowly in the gel because of extra negative charges. Instead they moved more rapidly, consistent with deacetylation. Finally, the H4 peptide shows no evidence for ADP-ribosylation in the HPLC chromatogram (Fig. 4B), as argued above.

Overproduction of SIR2 in yeast was earlier found to cause bulk deacetylation of histones, leading to the suggestion that SIR2 was a histone deacetylase or stimulated such an activity (18). The work presented here demonstrates that SIR2 can deacetylate histones, and is the likely explanation for the overexpression phenotype observed previously. At normal levels, SIR2 presumably deacetylates histones in chromatin where it is bound, including the silent mating loci, the telomeres, and within the rDNA. When SIR2 is removed by mutation, these loci are aberrantly activated, and recombination is observed to increase within the repetitive rDNA (reviewed in refs. 2 and 3).

We observed that HST2 and SIR2 can both deacetylate histones that have been acetylated by HAT1 or ESA1 (Fig. 3A). These two HATs have somewhat different substrate specificities and different biological roles. HAT1 is likely to function in chromatin assembly by modifying newly synthesized histones, thereby promoting their deposition in the nucleosomal octamer of histones (14, 19). In contrast, ESA1 is the only known essential HAT in yeast and may contribute to both transcriptional regulation and other critical biological processes (16, 17, 20). The fact that the recombinant SIR2-like enzymes can deacetylate histones modified by either class of HATs raises several possibilities. One is that the enzymes have very broad specificities in vivo, and may in fact have the capacity to modulate chromatin structure under many different circumstances to yield different biological consequences. A second possibility is that the functions of the family members are far more restricted in vivo, and the activities that we observe here reflect some relaxation of enzyme specificity. This would not be unprecedented and introduces the possibility for significant control mechanisms. For example, because SIR2 is targeted primarily to silenced loci, perhaps its activity is restricted to these loci and modulated by other components of silencing complexes. The recent observation that SIR2's localization changes during mitosis (5) raises the possibility that its activity may also be dynamic. Less is known about HST2, but because it does not function in mating type or telomeric silencing (7), it presumably functions elsewhere in the cell, perhaps in the context of a complex of proteins that contribute to its specificity. Indeed, reports of cytoplasmic localization for human and trypanosome homologs (21, 22) that are closely related to HST2 emphasize the necessity of thinking broadly about the roles of these enzymes.

The functions of the homologs of SIR2 in other organisms have not yet been well established. The fact that E. coli CobB has comparable catalytic activity to two eukaryotic enzymes suggests that not all homologs will function to deacetylate histones, and that there may be other significant in vivo substrates. For example, diverse proteins such as myoD, p53, and tubulin are known to be acetylated on specific lysine residues (23–25). Perhaps these proteins are deacetylated by SIR2-like proteins to modulate their biological activities. Indeed, mutation of a fission yeast SIR2 homolog results in sensitivity to microtubule-depolymerizing drugs and chromosome loss (26). Likewise, in the fungus Candida albicans, mutation of a SIR2 homolog leads to genomic instability, which is correlated with clinical pathology (27). Defects in acetylation status of the mitotic spindle, centromeric histones, or other protein substrates would be consistent with these phenotypes.

Recent studies indicate that, in human cells, hyperacetylation may be a determinant for productive V(D)J recombination that ultimately yields appropriate Ag receptor gene expression in differentiated B and T lymphocytes (28). Because human homologs of the SIR2 family of deacetylases are expressed in multiple tissues, including those of lymphocytic and erythropoietic lineages (9, 29), it seems possible that these enzymes may contribute to processes as diverse as suppression of recombination, maintenance of genome stability, and transcriptional regulation. Understanding the unique mechanism and targets of these NAD-dependent deacetylase activities will be critical goals for future studies.

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