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Summary: Base editors (BEs) are emerging tools used for precision correction or diversifying mutation. It provides a potential way to recreate somatic hypermutations (SHM) for generating high-affinity antibody, which is usually screened from antigen-challenged animal models or synthetic combinatorial libraries. By comparing somatic mutations in the same genomic context, we screened engineered deaminases and CRISPR-deaminase coupling approaches and updated diversifying base editors (DBEs) to generate SHM. The deaminase used in DBEs retains its intrinsic nucleotide preference and mutates cytidines at its preferred motifs. DBE with AID targets the same hotspots as physiological AID does in vivo, while DBE with other deaminases generates distinct mutation profiles from the same DNA substrate. Downstream DNA repair pathways further diversified the sequence, while Cas9-nickase restricted mutation spreading. Finally, application of DBE in an antibody display system achieved antibody affinity maturation ex vivo. Our findings provide insight of DBE working mechanism and an alternative antibody engineering approach. : Liu et al. developed diversifying base editors (DBEs) with multiple engineered AID/APOBEC deaminases and discovered deaminase’s intrinsic nucleotide preference as one key feature of DBEs. With an expanded DBE toolbox, diverse mutation profiles are generated on immunoglobulin transgenes and antibody affinity maturation is achieved in ex vivo cultured cells. Keywords: base editor, antibody affinity maturation, somatic hypermutation, AID, diversifying base editor, APOBEC, CRISPR, antibody diversification, SHM, deaminase

Pneumococcal cell wall polysaccharide (C-PS), a contaminant in pneumococcal capsular polysaccharide (Pn-PS) vaccines is degraded by mild deamination of the 4-amino-2-acetamido-2,4,6-tri-deoxy-galactose (AAT) in C-PS, which was carried out by addition of 5% aqueous sodium nitrite to a solution of polysaccharide in 5% aqueous acetic acid. Glycosidic linkage and functional groups such as O-acetates, phosphodiesters, and pyruvates were preserved under the conditions. The small fragments from degraded C-PS were removed by ultrafiltration or dialysis to provide essentially C-PS free Pn-PS. Because of the presence of AAT in its structure the deamination is not suitable for the purification of type 1 Pn-PS. Meanwhile, the mass and NMR spectroscopic analysis on the deamination products suggests that both type 1 Pn-PS and C-PS degraded following a major pathway of 5,4-hydride shift, cleavage of AAT O5-C1 bond, C1 hemiacetal formation, and its hydrolysis to release neighboring GalA- in type 1 Pn-PS and GalNAc(6-O-PCho)- in C-PS.

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes., This work was funded by the Junta de Andalucía (BIO-199); the Plan Nacionalde Investigación Científica, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014) and FEDER; and the Plan Nacional (SAF2016-79957-R). We thank Dr Carlos Robello for kindly providing the anti-TbTRYP1 antibody and Dr David Horn for the anti-TbγH2A antibody.

Anaerobic choline deamination catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as a major route for trimethylamine (TMA) production within anaerobic environments, including the human gut. The association of this microbial metabolite and its downstream products with diseases such as atherosclerosis and chronic kidney disease has driven the need for a better molecular understanding of TMA-generating enzymes. Our previous work has shown that generating the critical, glycine-centered radical species on CutC requires posttranslational modification by an S-adenosyl- l -methionine (SAM)-dependent radical-activating protein (CutD) harboring an oxygen-sensitive [4Fe–4S] cofactor. In this chapter, we describe our strategy to heterologously express and purify Desulfovibrio alaskensis G20 CutD in Escherichia coli and reconstitute its iron–sulfur center under anaerobic conditions. In addition, we present the methods we have developed to characterize the activity of CutD and utilize this enzyme in conjunction with purified CutC to gain an unprecedented insight into the anaerobic C–N cleavage of choline.

The cytochrome P450 monooxygenase enzymes (P450s) catalyze a diverse array of chemical transformations, most originating from the insertion of an oxygen atom into a substrate that binds close to the P450 heme. The oxygen is delivered by a highly reactive heme iron-oxo species (compound I) and, according to the chemical nature of the substrate and its position in the active site, the P450 can catalyze a wide range of reactions including, e.g., hydroxylation, reduction, decarboxylation, sulfoxidation, N- and O-demethylation, epoxidation, deamination, CC bond formation and breakage, nitration, and dehalogenation. In this chapter, we describe the structural, biochemical, and catalytic properties of the P450s, along with spectroscopic and analytical methods used to characterize P450 enzymes and their redox partners. Important uses of P450 enzymes are highlighted, including how various P450s have been exploited for applications in synthetic biology.

Endogenous and dietary nitrite produces reactive nitrogen species (RNS) that react with DNA causing mutations. The nitrosation of 2′-deoxyguanosine (dGuo) and DNA with nitrite was studied under different conditions, and the reaction and degradation products identified and analysed by HPLC-DAD-MS. Nitrosative deamination of dGuo produced xanthine along with 2′-deoxyxanthosine whereas DNA afforded xanthine. Formation of xanthine increased with nitrite concentration and in low pH such as that of stomach. Xanthine was measured as a marker of nitrosation of dGuo and DNA, and it was subsequently used to study the antinitrosating activity of β-carboline alkaloids, and selected antioxidants. Food-occurring tetrahydro-β-carbolines (THβCs) decreased nitrosative deamination of dGuo and DNA under conditions simulating the stomach. Antinitrosating activity was also evidenced for flavonoids (catechin, quercetin) and indole (melatonin) antioxidants. Among THβCs the most active antinitrosating compounds were 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids (THβC-3-COOHs) that reacted with nitrite to give N-nitroso derivatives as main products along with 3,4-dihydro-β-carboline-3-carboxylic acids and aromatic β-carbolines (norharman and harman). Antinitrosating activity of THβCs correlated well with the formation of N-nitroso-THβC-3-COOHs. These N-nitroso derivatives were stable at pH 7 but degraded in acid conditions affording nitrosating species., The authors thank MINECO-FEDER (projects SAF2015-66690-R and SAF2015-68580-C2-2-R) and CSIC (project 200470E658) for supporting this work.

Transitions of cytosine to thymine in CpG dinucleotides are the most frequent type of mutations observed in cancer. This increased mutability is commonly explained by the presence of 5-methylcytosine (5mC) and its spontaneous hydrolytic deamination into thymine. Here, we describe observations that question whether spontaneous deamination alone causes the elevated mutagenicity of 5mC. Tumours with somatic mutations in DNA mismatch-repair genes or in the proofreading domain of DNA polymerase ε (Pol ε) exhibit more 5mC to T transitions than would be expected, given the kinetics of hydrolytic deamination. This enrichment is asymmetrical around replication origins with a preference for the leading strand template, in particular in methylated cytosines flanked by guanines (GCG). Notably, GCG to GTG mutations also exhibit strand asymmetry in mismatch-repair and Pol ε wild-type tumours. Together, these findings suggest that mis-incorporation of A opposite 5mC during replication of the leading strand might be a contributing factor in the mutagenesis of methylated cytosine.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

The bicarbonate buffer system is important because the concentrations of its components are independently regulated by the lungs and kidneys. The total CO2 content of blood can be estimated from the Pco2 and the plasma bicarbonate (HCO3−) concentration. The hemoglobin and bicarbonate buffer systems are the most quantitatively important buffer systems in blood. The plasma ratio of H2PO4−:HPO4 = is normally about 1:4. Cellular organic phosphate esters exist mainly in the dianionic form at physiologic pH. Inorganic phosphates are important buffers in the renal tubular filtrate. Most NH4+ destined for urinary excretion is formed in proximal renal tubular epithelial cells from the deamination of glutamine. The ammonia buffer system plays an important role in the rumen and large intestine.

Transglutaminase (TG) modifies proteins by amine incorporation, crosslinking and deamination. It is used as an important tool in food processing. The aim of this work was to crosslink ovalbumin (OVA) and egg white (EW) with TG, either under high pressure (HP) or under atmospheric pressure on previously HP-treated proteins, to assess the allergenic potential of polymerized allergens. The susceptibility of OVA and EW to TG-mediated crosslinking was enhanced by HP (400 MPa, 40 °C, 30 min). Simultaneous or sequential HP and TG treatments led to the formation of high-molecular weight polymers, but left a substantial amount of monomeric proteins. Results of digestibility experiments, binding to immunoglobulin E (IgE) from egg allergic patients and stimulation of spleen and mesenteric lymph node cell cultures of EW-sensitized BALB/c mice showed that resistance to proteolysis, IgE-binding and immunostimulatory properties of the HP and TG-treated proteins were not substantially different from those of the native ones. [Industrial Relevance]: The use of transglutaminase as a crosslinking agent has been suggested for many industrial applications, as in the case of meat, fish, milk or soy proteins, which are good substrates for this enzyme. This work widens the possible use of transglutaminase, for modifying less suitable substrates for crosslinking, as egg proteins, through the use of high pressure before or during crosslinking. In our study, HP-assisted TG-induced OVA and EW crosslinking enhanced gastroduodenal digestion but did not reduce the allergenicity of egg proteins., Financial support was received from AGL2011-24740 project from MINECO (Spain). E. M, R. L-F and D. L-O are participants in the COST-Action ImpARAS FA1402. D. L-O is a recipient of an FPU fellowship from MECD (Spain). The author X.J. Ma thanks the financial support from the China Scholarship Council (CSC, 201306820013).

Amino acids released from dietary or intracellular proteins are catabolized in similar fashions. Their degradation begins with the removal of the a-amino group, thus leaving an a-keto acid hydrocarbon skeleton. Alpha-amino group removal from amino acids occurs through either transamination or deamination reactions. Transamination reactions are catalyzed by transaminases (also called aminotransferases). There are four coenzymes intimately involved in amino acid catabolism: pyridoxal phosphate (derived from vitamin B6), tetrahydrofolic acid (THFA), biotin (a CO2 shuttler), and vitamin B12 (cobalamin). The oxidative deamination of Glu, catalyzed by mitochondrial GLDH, results in the direct release of ammonium ion (NH4 +), which can then be used for mammalian urea synthesis in the liver. The side chain amide groups of glutamine (Gln) and asparagine (Asn) can be released by hydrolysis, also resulting in NH4 + formation. Glutaminase and asparaginase, respectively, catalyze these reactions. During starvation, Ala is important in transferring amino groups from skeletal muscle to liver, where they can be incorporated into urea or Gln, and the pyruvate hydrocarbons remaining can be incorporated into glucose. Aspartate Aminotransferase is present in both the cytosol and mitochondria of liver cells, and both fractions can be separated electrophoretically. Other transaminases also exist in mammalian organisms (e.g., cysteine transaminase, ornithine transaminase, and tyrosine a-ketoglutarate transaminase). Since there are no reported metabolic defects associated with reactions catalyzed by any of the transaminases, like reactions of the tricarboxylic acid cycle, all are considered essential for life.

tThe most common mutations in cancer are C to T transitions, but their origin has remained elusive.Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many com-mon cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism.Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows sig-nificantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancercell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AIDmRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdownsignificantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergoclass switch recombination displayed a several-fold increase in total genomic uracil, indicating that Bcells may undergo widespread cytosine deamination after stimulation. In line with this, we found thatclustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carryAID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil withuracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion,AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutationsin B-cell malignancies. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Incorporation of fluorine into antibiotics can moderate their biological activity, lipophilicity and metabolic stability. The introduction of fluorine into an antimicrobial lipopeptide produced by Bacillus sp. CS93 via precursor-directed biosynthesis is described. The lipopeptide surfactin is synthesised non-ribosomally by various Bacillus species and is known for its biological activity. Administering 4,4,4-trifluoro-dl-valine to cultures of Bacillus sp. CS93 results in the formation of trifluorosurfactin in quantities sufficient for detection by LC–MS/MS. 19F NMR analysis of the culture supernatant revealed that the bulk of the fluorinated amino acid was transformed and thus was unavailable for incorporation into surfactin. Detection of ammonia, and MS analysis indicated that the transformation proceeds with deamination and reduction of the keto acid, yielding 4,4,4-trifluoro-2-hydroxy-3-methylbutanoic acid. IRCSET Enterprise Partnership Author has checked copyright SB. 30/4/2013

In response to an assault by foreign organisms, peripheral B cells can change their antibody affinity and isotype by somatically mutating their genomic DNA. The ability of a cell to modify its DNA is exceptional in light of the potential consequences of genetic alterations to cause human disease and cancer. Thus, as expected, this mechanism of antibody diversity is tightly regulated and coordinated through one protein, activation-induced deaminase (AID). AID produces diversity by converting cytosine to uracil within the immunoglobulin loci. The deoxyuracil residue is mutagenic when paired with deoxyguanosine, since it mimics thymidine during DNA replication. Additionally, B cells can manipulate the DNA repair pathways so that deoxyuracils are not faithfully repaired. Therefore, an intricate balance exists which is regulated at multiple stages to promote mutation of immunoglobulin genes, while retaining integrity of the rest of the genome. Here we discuss and summarize the current understanding of how AID functions to cause somatic hypermutation.

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side-chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The enzymes of the riboflavin pathway are potential targets for antibacterial agents. This chapter will review structures and reaction mechanisms of enzymes involved in the biosynthesis of riboflavin up to 2008 with 215 reference citations.

Single sites of selective adenosine (A) to inosine (1) RNA editing with functional consequences on the proteome are rarely found in mammals. Here we describe a method that can be used to detect nov ...

Publisher Summary This chapter focuses on the nucleobase deamination in light of recent technical advances in analytical methods to quantify nucleobase deamination products, as well as several recent observations on the stability of these DNA lesions and the diverse mechanisms of their formation. It covers the numerous established and potential mechanisms by which nucleobase deamination products can arise in or enter DNA and RNA. Among all the types of DNA damage, nucleobase deamination appears to have the most abundant and diverse mechanisms of formation, including nitrosative deamination at sites of inflammation, simple hydrolysis, the actions of several deaminase enzymes on DNA and RNA and defects in purine and pyrimidine metabolism. This level of complexity suggests that nucleobase deamination products could act as biomarkers of several facets of cellular pathophysiology, while their involvement in carcinogenesis and other disease processes could involve both direct mutagenesis and, via the nucleotide pools, disruption of cell signaling, cycling or other facets of cell physiology.

Microbial transglutaminase, TGM, a protein-glutamine γ-glutamyltransferase catalyses acyl transfer reactions introducing covalent crosslinking between proteins, deamination of glutamine residues and amine incorporation, and is considered as a processing aid in regulatory status. The TGM-catalysed reactions modify the functional properties of foods via aggregation and polymerization of proteins and may produce beneficial effects during breadmaking that are comparable to traditional oxidizing improvers, hypothesized to act via formation of disulfide crosslinks. The effects of TGM when added singly and in binary and ternary combinations with amylolytic (α-amylase, NMYL) and non-amylolytic enzymes (xylanase, PTP) on the physico-chemical and sensory profile of fresh and up to 20 day-old pan breads have been investigated in 16 samples made with low and high extraction rate wheat flours following a sponge-dough process. TGM when added to NMYL and NMYL/PTP supplemented doughs, induced synergistic beneficial effects on fresh bread quality and staling kinetic parameters leading to breads with increased specific volume and crumb cohesiveness, softer fresh crumb and slower crumb firming kinetics and sensory deterioration during storage, particularly for samples made with white flours. For high extraction rate flour samples, NMYL addition gave the greatest benefits in fresh bread quality and staleness retardation, where TGM effects were not as relevant.

Publisher Summary The biotransformation of drugs is divided into two categories—Phase I and Phase II metabolism. Phase I reactions include oxidation, reduction, hydrolysis, and hydration. Metabolic oxidations usually occur through the action of cytochrome P450 (CYP) oxidative enzymes. Although there are at least 50 different P450 isoforms, drug metabolism in humans most likely involve CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6. P450 enzymes catalyze aromatic hydroxylation, aliphatic hydroxylation, N-, O-, and S-dealkylation; N-hydroxylation, N-oxidation, sulfoxidation, deamination,and dehalogenation. This chapter focuses on CYP enzymes for Phase I metabolism and UDP-glucuronosyltransferase (UGT) enzymes for Phase II metabolism. Understanding the involvement of P450 enzymes in drug metabolism is critical to assessing the potential for drug interaction with concomitant drugs, food, and endogenous substances. Phase I enzymes primarily modify lipophilic molecules by creating polar functionalities to increase hydrophilicity, which thereby facilitates clearance from the body. Such additional functionalities may be readily amenable to Phase II conjugation reactions. Glucuronidation is the major conjugation pathway probably due to the relatively high natural abundance of the reaction co-factor, UDP-glucuronic acid. This process occurs with alcohols, phenols, hydroxylamines, carboxylic acids, amines, sulfonamides, and thiols. The role of these enzymes in drug metabolism is reviewed within the context of their polymorphism and analytical technologies used today in determining their involvement in drug metabolism are presented.

Summary The origin of reactive α-dicarbonyl species formed during the Maillard reaction can be identified through labelling studies using 13C-labelled sugars and amino acids. Such studies could be performed conveniently by Py-GC-MS. Analysis of model systems consisting of short-chain dicarbonyls. D-glucose and labelled glycine; indicated that α-ketoaldehydes produced by retro-aldol reactions of sugar derivatives could undergo chain elongation through incorporation of amino acid carbons, by two processes; (1) aldol condensation with Strecker aldehydes or (2) aldol condensation with the α-carbon of the amino acids followed by deamination. The resulting α-carboxydicarbonyl, in turn, can either undergo decarboxylation to produce dicarbonyls that incorporate the side chain of the amino acids or undergo further additions of amino acids to produce pyrazinones.

Publisher Summary Ammonia is mainly produced in fish through catabolism of amino acids. Ingested proteins are hydrolyzed producing amino acids, the catabolism of which leads to the release of ammonia. Ammonia production takes place mainly in the liver in fish but other tissues are also capable of doing so. Ammonia is produced by the transamination of amino acids followed by the deamination of glutamate and by the deamination of adenylates in fish muscle during severe exercise. Most aquatic animals keep body ammonia levels low by simply excreting excess ammonia produced through digestion and metabolism. The dietary protein requirement of fish increases at higher temperatures, possibly due to increased oxidation of amino acids reviewed the effects of temperature on nitrogen metabolism and concluded that as temperature increased an increasing percentage of aerobic metabolism was fueled by oxidation of protein. That is, ammonia production increases with increasing temperature due to both increased metabolic rate and a switch to greater protein utilization. However, this conclusion was based on studies of temperate fishes and, therefore, may not be applicable to tropical fishes. Tropical fishes show a marked increase in food intake and ammonia excretion with increasing temperature but the effect on protein metabolism is not known.

The levels of ammonia in cherimoya (Annona cherimola, Mill) fruit changed in response to different storage conditions. The highest amount of ammonia was observed in fruit stored at a chilling temperature, and the lowest ammonia level was found in CO2-treated fruit at ambient temperature. These fruit presented the highest amount of nitrogen incorporated in polyamines, mainly in spermidine and spermine. Moreover, the fact that the trend in polyamines and ammonia content reverted when treated fruit were transferred to air would appear to confirm preferential reassimilation of ammonia under high CO2 treatment. The increase in phenylalanine ammonia-lyase (PAL) activity observed in ripening fruit at ambient temperature was substantially reduced in fruit stored at a chilling temperature. Our results suggest that PAL activity increases in fruit with a high demand of ammonia for further assimilation processes, and that this activity decreases in fruit where ammonia reassimilation does not take place. Additionally, in the case of CO2-treated fruit, ammonia availability is apparently promoted by the deamination of amino acids, which is reflected in a significant decrease in free amino acid content without a concomitant rise in total protein levels. We observed that the patterns of change in phosphoenolpyruvate carboxylase (PEPC) and PAL activities were opposite. Moreover, coordination between these enzymes was related to the metabolic endogenous levels of ammonia. The role of ammonia levels as a significant metabolic signal for controlling carbon and nitrogen metabolism is discussed. © 2002 Elsevier Science B.V. All rights reserved.

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. Two reaction steps in the biosynthetic pathway catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase and riboflavin synthase are mechanistically very complex. The enzymes of the riboflavin pathway are potential targets for antibacterial agents.

Publisher Summary This chapter discusses several of the new developments, in particular the diversity of BER mechanisms, the regulatory control of BER, and the mutation frequency observed for the BER process for the removal of uracil. One of the major lesions acted upon, by the BER pathway, is uracil. This base is introduced in DNA, by spontaneous deamination of cytosine or by incorporation of deoxyuridine monophosphate (dUMP) during DNA synthesis. Clearly, a uracil introduced opposite any base other than adenine would be a mutagenic event unless corrected and accordingly, a diminished capacity for base excision repair of uracil-modified DNA leads to a mutator phenotype. The chapter discusses the products of the complete BER carried out, by human cell-free extracts on a uracil, specifically, positioned in the M13mp2 plasmid.

The human UNG-gene at position 12q24.1 encodes nuclear (UNG2) and mitochondrial (UNG1) forms of uracil-DNA glycosylase using differentially regulated promoters, PA and PB, and alternative splicing to produce two proteins with unique N-terminal sorting sequences. PCNA and RPA co-localize with UNG2 in replication foci and interact with N-terminal sequences in UNG2. Mitochondrial UNG1 is processed to shorter forms by mitochondrial processing peptidase (MPP) and an unidentified mitochondrial protease. The common core catalytic domain in UNG1 and UNG2 contains a conserved DNA binding groove and a tight-fitting uracil-binding pocket that binds uracil only when the uracil-containing nucleotide is flipped out. Certain single amino acid substitutions in the active site of the enzyme generate DNA glycosylases that remove either thymine or cytosine. These enzymes induce cytotoxic and mutagenic abasic (AP) sites in the E. coli chromosome and were used to examine biological consequences of AP sites. It has been assumed that a major role of the UNG gene product(s) is to repair mutagenic U:G mispairs caused by cytosine deamination. However, one major role of UNG2 is to remove misincorporated dUMP residues. Thus, knockout mice deficient in Ung activity (Ung-/- mice) have only small increases in GC-->AT transition mutations, but Ung-/- cells are deficient in removal of misincorporated dUMP and accumulate approximately 2000 uracil residues per cell. We propose that BER is important both in the prevention of cancer and for preserving the integrity of germ cell DNA during evolution.

Publisher Summary Liver plays a role in the inactivation of circulating catecholamines through an enormous capacity for removal and metabolic degradation of these amines. The capacity of the liver to metabolically inactivate catecholamines is in agreement with the presence of huge amounts of the catecholamine-metabolizing enzymes (monoamine oxidase [MAO], catechol O-methyltransferase [COMT], aldehyde dehydrogenase and reductase, and conjugating enzymes) in this organ. The removal and metabolism of catecholamines were studied using rat freshly isolated hepatocytes, liver slices, and perfused liver. The removal and metabolism of [ 3 H] noradrenaline ([ 3 H] NA) and [ 3 H]-adrenaline ([ 3 H] AD) were studied using isolated hepatocytes. There was no accumulation of [ 3 H] amines in the cells, as the cell-medium ratio was about unity. For [ 3 H] AD, O-methylation predominated, because O-methylated and deaminated metabolites ([ 3 H] OMDA) and [ 3 H] MN were the most abundant metabolites. For [ 3 H] NA, deamination predominated: [ 3 H] OMDA and [ 3 H] DOMA were the most abundant metabolites. In experiments with [ 3 H]NA, simultaneous inhibition of MAO and COMT originated a strong reduction in the formation of all [ 3 H]metabolites, but there was no increase in the amount of [ 3 H]NA in cells. Other studies on the removal and metabolism of [ 3 H] NA have been mentioned. It is seen that the rat liver possesses a huge capacity to remove and metabolize AD and NA, being AD preferentially O-methylated and NA preferentially deaminated; there is no accumulation of catecholamines in the liver, even after simultaneous inhibition of MAO and COMT, possibly because of the existence of an active extrusion system; and the pattern of metabolism of catecholamines is dependent on the experimental model studied. For the hepatic uptake of catecholamines, the uptake systems for catecholamines uptake 1 and uptake 2 are not involved, the type-1 hepatic transporter of organic cations seems to play a significant role, and a P-glycoprotein transporter also seems to be involved, in freshly isolated rat hepatocytes.