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Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. Pyogenes utilizes Cas9 to remember and later probe and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. In case that the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In addition to its original role in bacterial immunity, the Cas9 protein has been heavily employed as a genome engineering tool to induce site-directed double strand breaks in DNA. These disruptions may lead to gene inactivation or the presentation of heterologous genes via non-homologous end joining and homologous recombination respectively in many laboratory model organisms.

Glycolate oxidase (HAO1) is a part of the superfamily of the alpha hydroxy acid oxidases (HAO) enzymes. HAO1 catalyses the FMN mediated oxidation of glycolate to glyoxylate and glyoxylate to oxalate by reducing oxygen to hydrogen peroxide. HAO1 is expressed mainly in the liver and pancreas and is most active on twocarbon substrates such as glycolate. HAO1 isthe main cause of hyperoxaluria, a disorder in which large deposits of calcium oxalate form kidney stones.

Thrombin enzyme (Activated Factor IIa) is an important clotting promoter that controls the transformation of soluble fibrinogen to insoluble active fibrin strands. Thrombin is a coagulation protein and a serine protease (EC 3.4.21.5) that catalyzes many coagulation-related reactions. Thrombin triggers factor-XI, factor-V, Factor-XIII and factor-VIII. Thrombin endorses platelet activation, using activation of protease-activated receptors on the platelet. As a result of its high proteolytic specificity, thrombin has become an important biochemical protein. The thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser) is widely used in linker regions of recombinant fusion protein constructs. After the purification of the fusion protein, thrombin is used to cleave between the Arginine and Glycine residues of the cleavage site, efficiently removing the purification tag from the protein of interest with a high degree of specificity.

Lipocalin-1 (LCN1) belongs to the lipocalin family of small secretory proteins. Lipocalins are extracellular transport proteins, which bind to various hydrophobic ligands. LCN1 protein is the principal lipid binding protein in tears and is overproduced in response to numerous stimuli including infection and stress. LCN1 is a marker for chromosome aneuploidy as well as an autoantigen in Sjogren's syndrome.

DAAO is a peroxisomal enzyme which uses FAD (flavin adenine dinucleotide) as a cofactor and oxidizes D-amino acids to the corresponding amino acids, producing ammonia and hydrogen peroxide. DAAO substrates include an extensive array of D-amino acids, but it is inactive on the naturally occurring L-amino acids. DAAO acts on a variety of D-amino acids especially on those having small hydrophobic side chains followed by those bearing polar, aromatic, and basic groups; however it doesn’t act on acidic amino acids. DAAO may be involved in acid base balance in the kidney or it could act as a detoxifying agent which removes D-amino acids accumulated during aging. DAAO regulates the neuromodulator D-serine level in the brain. DAAO is highly active towards D-DOPA. Creatinine inhibits the DAAO in uremia. DAAO may also have a role in the pathophysiology of schizophrenia, but not in bipolar disorder.

EXO2 is a 3'-to-5' exonuclease specific for small (primarily 5 nucleotides or less in length) single-stranded RNA and DNA oligomers. RNA Exonuclease 2 (REXO2) takes part in DNA repair, replication, and recombination, and in RNA processing and degradation. EXO2 is involved in resistance of human cells to UV-C-induced cell death via its part in the DNA repair process.

Angiotensin converting enzyme 2 or ACE-2 is an enzyme that is located in the cell membranes in different organs such as kidney, intestines, lungs, heart & arteries. ACE2 acts as an entry receptor of SARS coronaviruses & SARS-CoV-2.The coronavirus spike (S) glycoprotein is a class I viral fusion antigen bound to the external envelope of the virion that has a role in a crucial part in viral infection by identifying host cell receptors and starting fusion of the viral and cellular membranes. Couple of main domains in coronavirus S1 have been identified, the N-terminal domain and C-terminal domain. One or the other and/or both S1 domains acts as a receptor-binding domain. SARS-CoV + MERS-CoV equally use C-domain to attach their receptors.ACE2 is a type I transmembrane antigen with an extracellular N-terminal domain having the catalytic site and an intracellular C-terminal tail. ACE2 has a signal peptide, a transmembrane domain & a single metalloproteinase active site holds an HEXXH zinc-binding domain. ACE-2 takes part as a mono-carboxypeptidase which degrades Ang I to produce the nonapeptide Ang 1–9 and Ang II to create the heptapeptide Ang 1–7.

Dehydrogenase/reductase SDR family member 9 (DHRS9) functions as a homotetramer, which converts both 3-alpha-tetrahydroprogesterone (allopregnanolone) and 3-alpha-androstanediol to dihydroxyprogesterone and is believed to have a part in retinoic acid biosynthesis.

Set 7/9 is a histone methyltransferase (HMTase) that transfers methyl groups to Lys4 of histone H3, in complex with S-adenosyl-L-methionine (AdoMet). The methylation of lysine residues of histones plays a critical role in the regulation of chromatin structure and gene expression.
Acetylation, phosphorylation and methylation of the amino-terminal tails of histone are thought to be involved in the regulation of chromatin structure and function. The enzymes identified in the methylation of specific lysine residue on histones belong to the SET family with just one exception. Set7/9, unlike most other SET proteins, is exclusively a mono-methylase.

DTD2 is e member of the DTD family. DTD2 protein hydrolyzes D-tyrosyl-tRNA(Tyr) into D-tyrosine and free tRNA(Tyr) as a defense mechanism against a harmful effect of D-tyrosine.

Pyruvate Dehydrogenase Complex, Component X, also known as PDHX, encodes the E3 binding protein subunit of the PDH complex which contains 3 catalytic subunits. PDHX tethers E3 dimers to the E2 core of the pyruvate dehydrogenase complexes of eukaryotes. This specific binding is critical for a functional PDH complex.

Uracil DNA glycosylase (UDG), or uracil-DNA glycosylase 1, is a crucial enzyme found in all life forms, involved in repairing damaged DNA by specifically removing uracil bases that are misincorporated into DNA during replication or deaminated cytosine. In various organisms, UDG goes by different names, such as b2580, JW2564, EC 3.2.2.27, DGU, UNG15, HIGM5, EC 3.2.2, HIGM4, and UNG2. Here, we delve into the E. coli UDG, examining its structure, function, and applications in molecular biology.

Structure: The crystal structure of E. coli UDG has been extensively studied, revealing that it belongs to the uracil DNA glycosylase (UDG) superfamily. The E. coli UDG monomer has 229 amino acids with a molecular weight of 25 kDa. The protein has a beta-sheet-rich structure with an alpha-helix on one side and a groove on the other side that binds to DNA. The active site of E. coli UDG contains a conserved glutamic acid residue that acts as a catalytic base to facilitate the hydrolysis of the N-glycosidic bond between uracil and the sugar phosphate backbone.

Function: E. coli UDG plays a critical role in maintaining the integrity of the genome by preventing the accumulation of mutations that can arise from the incorporation of uracil into DNA. Uracil in DNA can occur spontaneously from the deamination of cytosine or can be incorporated during DNA synthesis when dUTP is used instead of dTTP. Unrepaired uracil bases can lead to DNA damage and genomic instability, possibly resulting in cell death or disease. E. coli UDG specifically recognizes and removes uracil bases from DNA, creating an abasic site that is further processed by other repair enzymes.

Antioxidant enzyme Glutathione S- Transferase (GST) is thought to do the primary cellular defense mechanism against reactive oxygen species. GST reduces lipid hydroperoxides through its Se-independent glutathione peroxidase activity. The enzyme also detoxifies lipid peroxidation end products such as 4-hydroxynonenal (4-HNE).
The soluble GST is a 26 kDa protein which occurs as a dimer in all aerobic organisms. Each monomer has two domains, one that binds GSH and is an /-structure similar to thioredoxin and the other, all helical, that binds the hydrophobic substrate. The GST -fusion protein expression system is a widely used recombinant protein expression system that allows a peptide or a regulatory protein domain to be expressed as a fusion to the C-terminus of Schistosoma japonicum GST. Fusion proteins also possess GST -enzymatic activity and can undergo dimerization similar to in vivo. The fusion protein can be purified via GST -affinity column chromatography. In most cases, the desired peptides or domains are removed from GST by applying a specific protease that recognizes and cleaves the linker between the protein domain and GST. The technique has been widely used to generate different kinds of proteins for crystallization, molecular immunology studies, the production of vaccines and studies involving protein-protein and protein-DNA interactions.

GARS is an (alpha)2 dimer which is a member of the class II family of tRNA synthetases. GARS is a glycyl-tRNA synthetase, one of the aminoacyl-tRNA synthetases which charge tRNAs with their cognate amino acids. GARS catalyzes the attachment of glycine to tRNA(Gly). In addition, GARS is able to produce diadenosine tetraphosphate (Ap4A), which is a universal pleiotropic signaling molecule required for cell regulation pathways, by direct condensation of two ATPs. GARS has been demonstrated to be a target of autoantibodies in the human autoimmune diseases, polymyositis or dermatomyositis.

Membrane-bound & Cytosolic forms of GST are encoded by 2 separate supergene families. These enzymes function in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress, by conjugation with glutathione. There are 8 different classes of soluble cytoplasmic mammalian GST: alpha, kappa, mu, omega, pi, sigma, theta and zeta. The GSTA1 is found in a cluster mapped to chromosome 6, and is highly expressed in the liver. GSTA1 protects the cells from reactive oxygen species.

Spermidine/Spermine N1-Acetyltransferase 2, also known as SAT2 catalyzes the acetylation of polyamines (Acetyl-CoA + an alkane-alpha,omega-diamine = CoA + an N-acetyldiamine).

AKR1A1 is part of the aldo/keto reductase superfamily, it catalyzes the NADPH-dependent reduction from a range of aromatic and aliphatic aldehydes to their related alcohols. AKR1A1 corresponds (65% identity) to aldose reductase, an enzyme that takes part in the pathogenesis of some diabetic and galactosemic complications. AKR1A1 is involved in the activation of procarcinogens, such as polycyclic aromatic hydrocarbon trans-dihydrodiols, and in the metabolism of various xenobiotics and drugs.

Myristate, a rare 14-carbon saturated fatty acid, is co-translationally attached by an amide linkage to the N-terminal glycine residue of cellular & viral proteins with various functions. N-Myristoyltransferase 1, also known as NMT1 catalyzes the transfer of myristate from CoA to proteins. NMT1 seems to be irreversible and is essential for full expression of the biologic activities of several N-myristoylated proteins, as well as the alpha subunit of the signal-transducing guanine nucleotide-binding protein, G protein.

Phosphoribosylaminoimidazole Carboxylase (PAICS) is an enzyme involved in nucleotide biosynthesis and particularly in purine biosynthesis. PAICS is a bifunctional enzyme containing phosphoribosylaminoimidazole carboxylase activity at its N-terminal region and phosphoribosylaminoimidazole succinocarboxamide synthetase at its C-terminal region. PAICS catalyzes the conversion of 5'-phosphoribosyl-5-aminoimidazole(AIR) into 5'-phosphoribosyl-4-carboxy-5-aminoimidazole (CAIR) as described in the reaction. PAICS catalyzes steps six and seven of purine biosynthesis.

DNA polymerase beta (POLB) is a member of the DNA polymerase type-X family. In eukaryotic cells, POLB performs base excision repair necessary for DNA maintenance, replication, recombination, and drug resistance. POLB has 2 separate domains; the larger is the polymerase domain itself, whereas a small basic N-terminal domain contains an AP lyase activity which excises the abasic sugar-phosphate residue at the strand break. POLB fills single nucleotide gaps in DNA produced by the base excision repair pathway of mammalian cells. POLB overexpression, as seen in some human tumors, could convene an increase in spontaneous mutagenesis.

Glycerol dehydrogenase (GldA) catalyzes the NAD-dependent oxidation of glycerol to dihydroxyacetone (glycerone). The GldA protein allows microorganisms to use glycerol as a source of carbon under anaerobic conditions. Furthermore, in E.coli GldA has an imperative role by regulating the intracellular level of dihydroxyacetone by catalyzing the reverse reaction, i.e. the conversion of dihydroxyacetone into glycerol. GldA possesses an extensive substrate specificity, due to its ability to oxidize 1,2-propanediol and to reduce glycolaldehyde, methylglyoxal and hydroxyacetone into ethylene glycol, lactaldehyde and 1,2-propanediol, respectively.

HPRT1 has a main part in the generation of purine nucleotides through the purine salvage pathway. HPRT1 primarily functions to salvage purines from degraded DNA to renewed purine synthesis. Therefore, it performs as a catalyst in the reaction between guanine and phosphoribosyl pyrophosphate to form GMP.

Glycerol-3-phosphate dehydrogenase 1-like protein (GPD1L) converts sn-glycerol 3-phosphate to glycerone phosphate. GPD1L is found in the cytoplasm, associated with the plasma membrane, where it binds the sodium channel, voltage-gated, type V, alpha subunit (SCN5A). Mutations in the GPD1L gene are the cause of SIDS (sudden infant death syndrome) and Brugada syndrome type 2 (an autosomal dominant tachyarrhythmia).

SORD enzyme is part of of the zinc-containing alcohol dehydrogenase family that is broadly expressed in kidney and in the lens eye. SORD enzymatically catalyzes the zinc-dependent interconversion of polyols, such as sorbitol and xylitol, to their respective ketoses.