An Overview on D-Amino Acids

Amino Acids DOI 10.1007/s00726-017-2459-5

REVIEW ARTICLE

An overview on d‑amino acids

Giuseppe Genchi1

Received: 19 April 2017 / Accepted: 26 June 2017 © Springer-Verlag GmbH Austria 2017

Abstract More than half a century ago researchers thought Keywords d-amino acid · Amino acid racemase · d-amino that d-amino acids had a minor function compared to acid oxidase · d-Asp · d-Ser l-enantiomers in biological processes. Many evidences have shown that d-amino acids are present in high concentration in microorganisms, plants, mammals and humans Introduction and fulfl specifc biological functions. In the brain of mammals, d-serine (d-Ser) acts as a co-agonist of the N-methyl- Proteins are the most abundant biological macromolecules d-aspartate (NMDA)-type glutamate receptors, responsible occurring in all cells and all parts of cells. All proteins, both for learning, memory and behaviour. d-Ser metabolism is from bacteria and from the most complex forms of life, are relevant for disorders associated with an altered function of composed of 20 amino acids, covalently linked through the NMDA receptor, such as schizophrenia, ischemia, epi- a peptide bond in a myriad of different combinations and lepsy and neurodegenerative disorders. On the other hand, sequences. From these 20 building blocks, all organisms can d-aspartate (d-Asp) is one of the major regulators of adult turn out different products such as enzymes, hormones, trans- neurogenesis and plays an important role in the develop- porters, antibodies, lens proteins, haemoglobin transporting ment of endocrine function. d-Asp is present in the neu- oxygen, cytochromes transporting electrons, antibiotics and a roendocrine and endocrine tissues and testes, and regulates myriad of other substances with distinct biological activities. the synthesis and secretion of hormones and spermatogen- All 20 standard amino acids found in proteins are esis. Also food proteins contain d-amino acids that are nat- α-amino acids, i.e. the general structure of the amino urally originated or processing-induced under conditions acids includes a carboxyl group and an amino group, both such as high temperatures, acid and alkali treatments and bonded to the α-carbon atom (the one next to the carboxyl fermentation processes. The presence of d-amino acids in group); this α-carbon is also linked both to a hydrogen and dairy products denotes thermal and alkaline treatments and to a side chain group. microbial contamination. Two enzymes are involved in the There are about 500 amino acids in nature, but only 20 metabolism of d-amino acids: amino acid racemase in the are proteogenic. Proteins are synthetized on polysomes in the synthesis and d-amino acid oxidase in the degradation. presence of mRNA, rRNA and tRNA as simple amino acid chains. Protein synthesis ends by a termination codon in the mRNA. To achieve its biologically active form, the new polypeptide must fold into its proper three-dimensional confor- mation after posttranslational reactions. The new polypeptide Handling Editor: J. D. Wade. can undergo enzymatic reaction, including the formation of * Giuseppe Genchi disulfde bridges; addition of methyl, carboxyl, acetyl, phos- [email protected] phoryl, palmitoyl, retinoyl, or other groups to some amino acid residues; proteolytic cleavage; attachment of oligosaccharides 1 Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria (UNICAL), Arcavacata or prosthetic groups. Amino terminus may be blocked, and dif- di Rende, Cosenza 87036, Italy ferent moieties, small and large, can be added to the reactive

1 3 G. Genchi groups of proteins with covalent modifcation reactions. As a Special nomenclature has been employed to specify the result of carbohydrates addition, biophysical properties as well absolute confgurations of the four substituents of asym- as biological activities and stability increase, interaction with metric carbon atoms. The absolute confgurations of simple membrane receptors or nucleic acids can change and enzymes sugar and amino acids are specifed by the l- and d-systems catalytic properties can depend on side chain modifcation. based on the absolute confguration proposed by Emil Fis- Besides, the reversible phosphorylation of serine, threonine cher (1891) of the three-carbon atom glyceraldehyde, the and tyrosine (Burnett and Kennedy 1954) is greatly important smallest sugar to have an asymmetric carbon atom. For for the entire network of intracellular signalling. A new type all chiral compounds, stereoisomers with a confguration of posttranslational reaction has been revealed about 50 years related to that of l-glyceraldehyde are designated L, while ago, i.e. the conversion of certain amino acids in peptides or stereoisomers related to d-glyceraldehyde are designated proteins from the l- to the d-confguration (Bevins and Zasloff D. 1990; Kreil 1997). According to the convention of E. Fischer, the mol- ecule of glyceraldehyde is written (with respect to the chiral carbon atom) with aldehyde group (–CHO) upward

Stereochemistry of α‑amino acids and methyl group (–CH3) downwards. The –OH group on the chiral carbon is left in the l-stereoisomer and right in Every object has a mirror image and the elements of the pair the d-stereoisomer (Fig. 1). The amino acid alanine will of objects, that are mirror images, can be superimposed on be written with the carboxyl group (–COOH) upward, and each other. In other cases, the mirror image objects cannot the methyl group (–CH3) downward. The –NH2 group on be superimposed on each other, but are related to each other the chiral carbon is toward the left in the l-stereoisomer as the right hand is to the left hand. Such not-superimpos- and to the right in the d-stereoisomer (Fig. 1). It is impor- able mirror images are said to be chiral (from the classic tant to note that not all l-amino acids are L levorotary Greek “χειρ”, hand). Frequently a chiral centre in biomole- (rotating plane-polarized light to the left), but they can cules is a carbon atom (sp 3 hybridized) linked to four differ- rotate the plane of polarized light to the right; and in the ent substituents. For all standard amino acids, the α-carbon same way not all d-amino acids are D dextrorotary (rotat- is bonded to four different groups: a carboxyl group (– ing plane-polarized light to the right), but they can rotate

COOH), an amine group (–NH2), a hydrogen (–H) and a the plane of polarized light to the left. By E. Fischer’s side chain R group. Glycine has on α-carbon two hydrogen atoms; therefore, only glycine does not have a chiral centre. Because of the tetrahedral nature of sp3 orbitals of the carbon atom, the four different substituent groups can occupy two different spatial arrangements that are not superimposable mirror images to each other. These two forms, called enantiomers, represent a new class of stereoisomers. Isoleucine and threonine have four stereoisomers, because these amino acids have a second stereo- genic centre in their β-carbon atom. All molecules with a chiral centre are optically active and can rotate the plane- polarized light, when examined in a polarimeter. Optical activity is given by all compounds existing in two forms, whose structures are not superimposable mirror images to each other. This condition is met by compounds containing one (or more) asymmetric tetrahedral carbon atom(s), i.e. carbon atom(s) with four different substituents. The substances that rotate clockwise (to the right) to the plane of polarized light are said to be dextrorotatory (from the Latin “dexter”, right), while those that rotate counter- clockwise to (to the left) the plane of polarized light are said to be levorotatory (from the Latin “laevus”, left). In general, d- and l-stereoisomers (enantiomers) have the same chemical and physical properties, with the exception of the rotation of the plane of polarized light in dif- Fig. 1 Relationship of stereoisomers of alanine with the absolute ferent directions, i.e. dextrorotatory or levorotatory. confguration of l- and d-glyceraldehyde

1 3 An overview on d-amino acids convention, l and d refer only the absolute confguration bacterial activity produce high quantity of d-Ala in fruit of the four substituents around the chiral atom. juices (Gandolf et al. 1994). The observation by several authors that processed commercial foods contained various d-amino acids has prompted numerous studies investigating the presence of d‑Amino acids of dietary origin d-amino acids in a variety of foods. Gobbetti et al. (1994) found that the use of lactic acid and yeast in the fermen- Food proteins usually contain l-α-amino acids, but some tation of sourdough before baking results in the produc- d-isomers occur in food either naturally originated or tion of free d-Ala and d-Glu in the dough. processing-induced under specifc conditions such as In ewes’ and cows’ milk, d-amino acids originated from high temperatures, strong acid and alkali treatments, enzymatic digestion of peptides and proteins containing fermentation processes or cases of non-fermented foods d-amino acids derived from peptidoglycan proteins of adulteration (Hayase et al. 1975; Friedman et al. 1984; microbial cell walls in the ruminants’ rumen. In fact, milk Chiavaro et al. 1998). from cows, goats and ewes, but not human milk, con- Food stores prepare and sell increasing quantities of tains free d-Ala, d-Asp, d-Glu, d-Ser and d-lysine (d-Lys) foods (baked potatoes, fruit juices and fruit pulp, break- (Albert et al. 2007). In addition, these same amino acids fast cereals, tomato sauces, milk, etc.), which in some are present in daily consumed ripened cheeses, and the cases contain substantial quantities of d-amino acids d-amino acid content varies among cheeses and changes (Csapò et al. 2009). In these foods, the racemization pro- during cheese production (Pearce et al. 1988). cess is responsible for the formation of d-amino acids. The amino acids d-Ala, d-Asp, d-arginine (d-Arg) and The principal factors infuencing racemization are alka- d-Glu are present in fruits such as apples, grapes and line and acid pHs, treatment duration, heat treatment and oranges, and also in vegetables such as carrots, tomatoes, duration of heating. Milk, meat and fruit juices, which do cabbages as well as in the corresponding juices (Gandolf not contain substantial quantities of d-amino acids, are et al. 1994; Simó et al. 2004). It is unclear whether these often exposed, in the course of preparation for consump- d-amino acids could originate from plant sources, from tion, to conditions which may give rise to racemization. microorganisms present in the soil or from heat treat- Milk and dairy products serve as examples of how ments used to destroy pathogens. The presence of specifc the composition of natural substances can change (Bada d-amino acids could be used to differentiate juices from 1984). Most dairy products, for example, milk, are frst biologically dissimilar fruits and as an indicator for detect- pasteurized (involving heating for 2–3 min at 75–85 °C) ing bacterial activity of fruit juices (Friedman 2010). or ultra-pasteurized (involving heating for very short A delicacy of traditional Chinese cuisine is repre- period of 1–2 s at 135–145 °C). They are subsequently sented by pidan. Duck or chicken eggs are immersed subjected to homogenization and condensation, until a at room temperature for at least 30 days in an alkaline particular product, such as milk for commercial con- solution prepared with 4.2% NaOH and 5% NaCl, which sumption, yoghurt and cheese, derived from the various leads to extensive racemization of all ovalbumin l-amino milk protein fractions, is fnally obtained; yoghurt and acids in d-amino acids with the concurrent formation of cheese are fermented by means of bacteria, and this pro- lysinoalanine (Chang et al. 1999). cess also constitutes a source of d-amino acids. When we consider the taste of foods, d-amino acids have The presence of d-amino acids in dairy products can be sweeter taste compared to l-stereoisomers which generally used as a biomarker of thermal and alkaline treatments, and have bitter favour. In some cases, the sweetening power of as adulteration or fortifcation of the products. A concentra- d-valine (d-Val), d-phenylalanine (d-Phe) and d-tryptophan tion above 4% of d-alanine (d-Ala) in milk can represent (d-Trp) is higher than that of sucrose (Linden and Lorient microbial milk contamination (Oancea and Formaggio 1999). Alitame, an artifcial dipeptide sweetener contain- 2008); in wines and vinegar the presence of d-proline (d- ing l-Asp and d-Ala, is of commercial interest because it Pro) is used as an indicator for age dating (Chiavaro et al. is about 200 times sweeter than sucrose and about 10 times 1998); fermented milk products contain high amounts of d- sweeter than aspartame (Chattopadhyay et al. 2014). Ala, d-Asp and d-glutamic acid (d-Glu) (Csapò et al. 2006). Resulting from different microorganism activity, d-amino acids are quite common in diary foodstuffs Amino acid racemase and fermented beverages (wine, beer and vinegar). The use of bacteria and yeast in fermentation of sourdough, Two enzymes are chiefy involved in synthesis and degra- honey and liquid spices produce variable amounts of free dation of d-amino acids: amino acid racemase and d-amino d-amino acids (Csapò et al. 2006). Heat treatments and acid oxidase (DAAO). The enzyme racemase catalyses the

1 3 G. Genchi conversion between l- and d-amino acids and can be pyri- racemase. An essential cysteine residue is present in the doxal 5′-phosphate (PLP) dependent and PLP independent enzyme active site (Yamauchi et al. 1992). (Yoshimura and Esak 2003). In mammals, the enzyme serine racemase (Srr) converts Since l-amino acids are the predominant amino acids l- to d-Ser in the presence of PLP (Fig. 2), Mg++ and ATP found in living organisms proteins, they act as the sub- (Wolosker et al. 1999; De Miranda et al. 2002). Also Ca++ strate to generate d-amino acids. l- to d-amino acids or Mn++ was necessary for enzyme activity, whereas the occur by a reaction in the presence of the enzyme race- presence of chelators such as ethylenediaminetetraacetic mase that changes the stereochemistry of the chiral acid (EDTA) completely inhibited the enzyme serine race- α-carbon in amino acids. mase (Cook et al. 2002). By contrast, glycine, l-aspartic Alanine racemase is a PLP-dependent enzyme that acid and l-asparagine competitively inhibit this enzyme deprotonates and reprotonates on the opposite side the (Dunlop and Neidle 2005). Serine racemase is also strongly α-carbon of l-Ala generating d-Ala. The presence of a inhibited by reagents that react with sulfhydryl groups such lysine residue in the enzyme active site is essential for as glutathione. In addition, serine racemase converts d- to the mechanism of action of this enzyme (Watanabe et al. l-Ser albeit with lower affnity (Wolosker et al. 1999). 1999). d-Glu is a component of the peptidoglycan cell Many aspects regulating d-Ser production under physi- wall in bacteria; d-Glu is produced by a PLP-independent ological and pathological conditions are to be elucidated. glutamate racemase (Choi et al. 1992) with two cysteines In the Wolosker lab, Kolodney et al. (2015) investigated the involved in the catalysis. In addition, d-Asp occurs in mechanisms that regulate the synthesis of d-Ser by serine the peptidoglycan layer of same bacterial cell walls and racemase in paradigms relevant to neurotoxicity. Kolodney is produced from l-Asp in the presence of aspartate in his paper reports that serine racemase undergoes nucleo- racemase, an enzyme PLP independent as glutamate cytoplasmic shuttling and that this process is dysregulated

H - - HOH2C COO HOH2C C COO C + HO HNH + HN C H C H C P P P OH - OH OH + HOH2C C COO + + NH + N CH 3 N CH3 N CH 3 H+ 3 H H H L-serine Schiff base

H - HOH C COO - 2 C HOH C C COO 2 HO + + C H HN H HNH C C P - A OH HOH C C COO P P 2 OH OH + + + NH3 + N CH3 + N CH3 H N CH3 H H H D-serine

H - - H2C COO HOH2C C COO C + + HO HNH H O C 2 HNH - - C C P COO OH H2C COO H3C P P C NH + OH OH + C + 4 B + + NH O + + N CH3 3 N CH3 N CH H2O 3 H H H

Fig. 2 Serine racemase pyridoxal-5′-phosphate dependent. This duce iminopyruvate, which non-enzymatically hydrolyses to form enzyme catalyses a the racemization between l-serine and d-serine pyruvate and ammonia and b the α,β-elimination of water from l-serine or d-serine to pro-

1 3 An overview on d-amino acids by several insults leading to neuronal death by apoptotic neurodegenerative disorders; Fuchs et al. 2005), d-amino stimuli. In this way, cell death induction promotes nuclear acids could have negative effects in diet, frst of all affect- accumulation of serine racemase, nuclear translocation of ing the digestibility of food protein and the availability of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) the other l-amino acids (Man and Bada 1987). In fact, and seven in absentia homolog (Siah) proteins (that func- the peptide bonds with d-amino acids are more resistant tion as E3 ubiquitin ligase in proteins degradation) at the to proteases. This process results in a reduction of the early stage of cell death process. As a consequence of this quantity of the essential l-amino acids, since the peptide apoptotic insult, nuclear serine racemase and GAPDH are bonds cannot split in the normal way. completely inactivated and this implies that extracellular d- DAAO was frst described by Krebs (1935). This Ser concentration is drastically reduced, while extracellular enzyme is widespread in nature from microorganisms to glutamate concentration increases several times. invertebrates, plants, vertebrates and mammals. The mito- Human beings can acquire d-amino acids through inges- chondrial enzyme DAAO is an FAD-dependent enzyme tion of food, derivation from endogenous microbial fora, that catalyses the oxidative deamination of d-amino acid, liberation from metabolically unstable polypeptides, con- yielding hydrogen peroxide (H 2O2) and an imino acid taining d-amino acids after racemization with ageing, and (Fig. 3). This latter is non-enzymatically hydrolyzed to through biosynthesis from l-amino acids. an α-ketoacid and ammonium (NH 4+). The α-ketoacids Foltyn et al. (2005) and De Miranda et al. (2002) found can undergo a transamination enzymatic reaction PLP that serine racemase catalyses also the α, β-elimination of dependent, which results in the l-enantiomer of the origi- water from both l- and d-Ser producing pyruvate and ammo- nal amino acid, which in turn enters the usual metabolic nia (Fig. 2). Pyruvate is further metabolized by pyruvate processes, or alternatively is broken down in another dehydrogenase complex to acetyl-CoA and by Krebs cycle. reaction, like oxidative decarboxylation. A characteristic d-Asp is located in the nervous and reproductive systems of all the DAAO is their high specifcity towards d-amino with various physiological roles. Whereas several lines of acids, while they are inactive towards the corresponding evidence suggest that this amino acid has an endogenous l-amino acids. DAAO exhibits optimal activity towards origin, the enzyme responsible for mammalian d-Asp free neutral d-amino acids, and marginal activity towards biosynthesis has not yet been identifed. Ito et al. (2016) basic ones. Acidic amino acids are oxidized by another showed that mammalian enzyme serine racemase, the pri- favo-enzyme, d-aspartate oxidase. mary enzyme responsible for brain d-Ser production also DAAO is used as a bio-catalyst in several biotechno- catalyses Asp racemization. The authors observed that logical applications, such as the oxidation of cephalo- overexpression of serine racemase in rat pheochromocy- sporin C, and as the biological component in several bio- toma PC12 cells resulted in an increase in intracellular d- sensors for the determination of the content in d-amino Asp compared with control cells, demonstrating that this acids of biological fuids. To determine d-Ser levels in enzyme functions as an Asp racemase (Ito et al. 2016). the central nervous system in vivo, Pernot et al. (2008) developed a microbiosensor based on cylindrical plati- num microelectrodes, covered with a membrane of poly- d‑Amino acid oxidase (DAAO) m-phenylenediamine and a layer of immobilized DAAO from the yeast Rhodotorula gracilis. When implanted Despite of their benefcial role in some diseases in the cortex of anesthetized rats, this microbiosensor (for example, d-Ser in schizophrenia, epilepsy and detected the increase in concentration of d-Ser resulting

NH O NH2 DAAO H2O R COOH + NH3 R COOH R COOH

H FAD FADH2 aminoacid iminoacid α-ketoacid

H O 2 2 O2

Fig. 3 Scheme of the reaction catalysed by the d-amino acid oxi- in the presence of O2 producing FAD and H2O2. The imino acid is dase (DAAO). The d-amino acid is oxidized to an imino acid in the non-enzymatically hydrolyzed to the corresponding α-ketoacid and presence of the enzyme d-amino acid oxidase. FAD (favin adenine ammonia nucleotide) is reduced to FADH 2. In turn, FADH 2 is re-oxidized

1 3 G. Genchi from its diffusion across the blood–brain barrier after an In some marine worms and invertebrates, the cellular intraperitoneal injection. This device will make it possi- fuid contains d-amino acids as a main component (Corri- ble to investigate in vivo the variations in d-Ser concentra- gan 1969; D’Aniello and Giuditta 1978; Matsushima et al. tions occurring under normal and pathological conditions. 1984; Felbeck 1985); in certain marine shellfsh quantities of d-amino acids can exceed 1% (Preston 1987). Also higher plants contain d-amino acids (Robinson 1976). d- d‑Amino acids in living organisms Asp, d-Ala, d-Asn, d-Glu, d-Gln and d-Ser occur naturally in free state as in pea seedlings, tobacco leaves, wild rice Three different hypotheses have been formulated to explain and lentils (Brückner and Westhauser 1994). the presence of d-amino acids in living organisms’ proteins. Numerous studies have shown that the secretion of the It may result from different mechanisms such as direct skin of amphibians contains a large variety of biologically incorporation in the peptide chain of a d-amino acid (pro- active peptides, which have proved to be homologous or duced for example by amino acid racemase), non-enzy- identical to mammalian hormones and/or neurotransmit- matic racemization associated with ageing or diseases, and ters. After isolation of enkephalins from the human brain, enzymatic posttranslational modifcation. In the last case, Erspamer began to search for similar peptides in the skin the peptides containing d-amino acids are synthetized via of many amphibians. In the lab of Erspamer, Montecucchi a ribosome-dependent manner, and a normal codon for et al. (1981) isolated from the skin of tree frog Phyllome- l-amino acid is present in the mRNA at the position where dusa sauvagei the dermorphin, a heptapeptide containing the d-amino acid is processed in the biologically active d-Met, which binds with high affnity to μ-type opioid peptide by peptidyl aminoacyl l-d isomerization (Ollivaux receptors (Table 1). Erspamer et al. (1989, 1993) studied et al. 2014). the deltorphin, a linear heptapeptide present on the skin of Free d-amino acids and peptides containing d-amino tree frog Phyllomedusa bicolor containing d-Ala, which acids have been isolated from a great variety of organisms. acts as potent hallucinogen. This peptide shows an high For example, bacterial cell wall peptidoglycans contain d- affnity for δ receptors (Table 1). These peptides have the

Asp, d-asparagine (d-Asn), d-Glu, d-glutamine (d-Gln) and sequence Tyr-d-Xaa-Phe-Asp(or Glu)-Val-Val-Gly-NH2, d-Ala (Reaveley and Burge 1972; Bada et al. 1983; Csapò where d-Xaa is d-Met for dermorphin and d-Ala for deltor- and Henics 1991), playing an important role in the bacte- phin. Research carried by Jimenez et al. (1996) showed that ria resistance to proteolytic digestion (Tipper and Wright an octapeptide found in the venom of the fsh-hunting snail 1979). In Gram-negative bacteria, a single layer of pepti- Conus radiatus contains a d-Trp residue (Table 1). doglycans is suffcient to maintain the mechanical stability In humans, d-amino acids are considered physiologi- of the cell, while in Gram-positive bacteria, which lack an cally active compounds and markers of diseases, deriving outer cell membrane, the cell wall is thicker because of sev- from racemization of l-isomers (Hamase 2007). Wolosker eral layers of peptidoglycans. d-Ala and d-Glu (Cava et al. et al. (1999) have shown that d-Ser plays an important 2011) are the most common amino acids present in the role in both physiological and pathological processes in bacterial cell wall; however, in the peptidoglycans of some cerebral cortex. In other cases, proteins containing d-Asp bacteria are present other d-amino acids such as d-Asp and are formed owing to ageing of certain tissues, includ- d-Ser (Veiga et al. 2006). ing teeth (Helfman and Bada 1976), bone (human male

Table 1 d-Aminoacid in eukaryotic peptides

Drug d-Aminoacid Source Activity

ω-Agatoxin d-Ser Venom of funnel-web spider (Agelenopsis aperta) Blocks sodium channels Bombinins d-Allo-Ile Skin secretion of frogs (Bombinatoridae) Antimicrobial and hemolytic activity Contriphans d-Trp Venom of core snails (Conus radiatus) Causes tremor and mucous secretions when injected into fsh Deltorphins d-Ala Skin secretions of three frogs (Phyllomedusa bicolor) Binds to δ-type opiate receptors, acting as a hallucinogen Dermorphins d-Met Skin secretions of three frogs (Phyllomedusa sauvagi) Binds to μ-type opiate receptor and acts as an analgesic, more powerful than morphine Achatin I d-Phe Ganglia and atrium of African snail (Achatina fulica) Excitatory neurotransmitter controlling muscle contrac- tion Fucilin d-Asn Ganglia of African snail (Achatina fulica) Excitatory neurotransmitter controlling penis contractions

1 3 An overview on d-amino acids femur) (Ohtani et al. 1998) and eye cataracts (Fujii et al. species of mammals, d-Ser is especially localized to the 1999). frontal brain areas, such as hippocampus, hypothalamus In our life, d-amino acids may have important biologi- and striatum (Hashimoto et al. 1995), while the amount of cal effects. First of all, they may be enzymatically con- this d-enantiomer is small in cerebellum, medulla oblongata verted to l-amino acids by DAAO, and thus they provide and spinal cord (Hashimoto et al. 1995). In neonatal rats and a pool for amino acids necessary for synthesis of proteins mouse cerebellum, a high amount of d-Ser is present, which and for anaplerotic reactions of the Krebs cycle (Asp and decreases with age; throughout the life span d-Ser remains Glu). d-Amino acids may act antagonistically to l-amino present in the frontal brain areas (Hashimoto et al. 1995). acids thanks to deactivation the binding to a biological site Studies in vivo and in vitro have suggested that d-amino (Friedman and Levin 2012). acids and especially d-Ser have an important role in N-methyl- d-Amino acids play roles in human physiology and d-aspartate (NMDA) receptor-mediated neurotransmission pathology. The most abundant d-amino acids in mammals (Shleper et al. 2005; Wolosker et al. 2008). The NMDA are d-Ser and d-Asp. Fuchs et al. (2005) have studied the receptors are involved in important functions in physiologi- presence of these two amino acids in the central nervous cal and pathophysiological processes, such as learning and system. d-Ser has an important role as neuromodulator of memory (Collingridge 1987), synaptic formation (Danysz glutamatergic neurotransmission (Miller 2004; Bauer et al. and Parsons 1998), epilepsy (Giraldez and Girardi 2000) and 2005; Fuchs et al. 2005) and is also present in the verte- nociception (Palazzo et al. 2002). The NMDA receptor has brate retina (Estevens et al. 2003). d-Asp has a benefcial several modulation sites, where both glycine and d-Ser bind role for reproduction (D’Aniello et al. 2005) and is impli- with high affnity (Schell et al. 1997a). The naturally occur- cated in neuroendocrine functions (D’Aniello 2007). d-Ser ring d-Ser is localized in close vicinity to NMDA receptor, and d-Ala have been implicated or implied in pathophysiol- while glycine is differently distributed from d-Ser and the ogy of Alzheimer’s disease (Hamase et al. 2002). NMDA receptors (Hashimoto and Oka 1997; Schell et al. d-Ser and d-Asp are synthesized by serine racemase 1997a). d-Ser serves as a co-agonist of the NMDA receptor and aspartate racemase and are, respectively, degraded by in mammalian brains, and its behaviour is probably related DAAO and DDO. The presence of d-amino acids in physi- to neurological disorders such as Alzheimer’s disease and ological fuids is infuenced by age, diet, physiological state amyotrophic lateral sclerosis. Mothet and co-authors showed and antibiotic therapies. It is known, in fact, that peptides that depletion of d-Ser in the presence of DAAO attenuates containing natural and synthetic d-amino acids are used as NMDA receptor-mediated neurotransmission, demonstrating antibiotics. These antibiotics include gramicidin, bacitra- that d-Ser has an important role in binding to glycine site of cin, actinomycin, valinomycin, tyrocidine and many others. the NMDA receptor (Mothet et al. 2000). These antibiotics act by disrupting membranes of bacterial A number of evidences suggest that hypofunction of cell through ion channel formation (Chattopadhyay and glutamatergic neurotransmission via the NMDA receptor Kelkar 2005). plays a crucial role in the pathophysiology of schizophrenia To determine d-amino acids in mammals, sensitive and (Krysta et al. 1999; Hashimoto 2006). d-Ser, synthesized, selective methods are needed, such as gas chromatogra- as already mentioned, from l-Ser by serine racemase is an phy, high-performance liquid chromatography, high-per- endogenous co-agonist of the NMDA receptor (Wolosker formance capillary electrophoresis (Zhao et al. 2001) and et al. 1999). It is important to remember that studies with enzymatic activities (Hamase et al. 2002). With regard to serine racemase (Srr) knockout mice showed that levels of d-amino acid-containing peptides, mass spectrometry is d-Ser in the forebrain of Srr knockout mice are 80–90% another option to identify these amino acids (Koehbach lower than in wild-type mice (Horio et al. 2011). This result et al. 2016). explains that d-Ser production in forebrain is dependent on serine racemase activity. Several studies have shown that disturbed NMDA recep- Presence and function of d‑Ser in mammals tor neurotransmission, due to decreased d-Ser levels, is a factor that causes schizophrenia (Wang et al. 2001; Coyle First in 1992, Hashimoto et al. (1992) found d-Ser in the rat and Tsai 2004; Hashimoto 2006; Ferraris and Tsukamoto frontal brain area, representing about 20–25% of the total 2011; Labrie et al. 2012). Therefore, treatment with d-Ser amount of serine (d-Ser plus l-Ser). Subsequently, naturally is benefcial to reduce positive, negative and cognitive occurring d-Ser was found not only in the brain (Hashimoto symptoms in patients with schizophrenia (Tsai et al. 1998). et al. 1992; Hamase et al. 1997; Hashimoto and Oka 1997), As the NMDA receptor is related to some important physi- but also in peripheral tissue (Hashimoto et al. 1992; Hashi- ological and pathological processes, it has triggered the moto and Oka 1997; Sakai et al. 1998) and physiological idea of using d-Ser as a therapeutic drug, especially for the fuids (Hashimoto et al. 1993). Within the brain of various treatment of schizophrenia.

1 3 G. Genchi

But before considering d-Ser a therapeutic agent, it d-Ser is an endogenous ligand for NMDAR gener- would be necessary to solve the problem of its bioavail- ated from l-Ser by the enzyme serine racemase. Neuronal ability. When d-Ser is orally administered, DAAO imme- and glial localizations have been reported for both d-Ser diately metabolizes this d-amino acid, diminishing its bio- and serine racemase. GAPDH is an exclusively astrocytic availability. Therapeutic levels of d-Ser could be achieved enzyme that catalyses the frst committed step of l-Ser if d-Ser is co-administered with DAAO inhibitors (Ferraris biosynthesis. Ehmsen et al. (2013), using transgenic mice and Tsukamoto 2011; Sacchi et al. 2013; Hin et al. 2016). expressing enhanced green fuorescent protein under the Antipsychotic agents targeting dopamine D2 receptors are serine racemase promoter and mice with targeted deletion generally used for the treatment of schizophrenia. There- of serine racemase or GAPDH, demonstrate predominantly fore, a new antipsychotic agent has to be proposed as a new the neuronal sources of d-Ser dependent on astrocytic sup- strategy. In fact, d-Ser administration to laboratory rodents ply of l-Ser (Ehmsen et al. 2013). inhibits behaviours similar to schizophrenia induced by phencyclidine, a psychotomimetic agent (Contreras 1990). d-Ser is present at very high levels in the mammalian brain and at a much lower concentrations in the peripheral Presence and function of d‑Asp in mammals tissues. d-Ser is a physiological endogenous co-agonist at the glycine site of N-methyl-d-aspartate (NMDA) subtype d-Asp is an endogenous amino acid present in invertebrates of glutamate receptors. d-Ser is synthesized by pyridoxal- and vertebrates and plays an important role in nervous and 5′-phosphate-dependent serine racemase. d-Ser is also neuroendocrine system, as well as in the development of essential for neurotransmission, synaptic plasticity and the nervous system. d-Asp also acts as a neurotransmit- behaviour. d-Ser may also trigger NMDA-mediated neu- ter/neuromodulator; indeed, this d-amino acid has been rotoxicity and its deregulation may play a role in neurode- detected in synaptosomes and in synaptic vesicles, where generation. d-Ser action has not been previously compared it is released after chemical (K + ion, ionomycin) or elec- with that of endogenous glycine, and the relative impor- tric stimuli. In the endocrine system, d-Asp is involved in tance of the two agonists remains unclear. the regulation of hormone synthesis and release. In the rat Shleper et al. (2005) investigated the effciency of these hypothalamus, it enhances gonadotropin-releasing hor- two agonists in mediating NMDA receptors’ neurotoxic- mone’s (GnRH) release and induces oxytocin and vaso- ity in hippocampal slices. The authors report that removal pressin mRNA synthesis. In the pituitary gland (D’Aniello of endogenous d-Ser from slices by pretreating the tis- 2007), d-Asp stimulates the secretion of the prolactin sue with the enzyme d-Ser deaminase virtually abolished (PRL), luteinizing hormone (LH) and growth hormone NMDA-elicited neurotoxicity, but did not protect against (GH). In addition in the testes, it is present in Leydig cells kainate. Although endogenous glycine was ten times more and is involved in testosterone and progesterone release. concentrated than d-Ser, glycine is ineffective in mediating In neonatal and adult rats, d-Asp is predominantly local- NMDA receptor neurotoxicity. The effect of endogenous ized in endocrine glands, such as testes (D’Aniello et al. glycine could be observed by removing endogenous d- 1996; Schell et al. 1997b; Sakai et al. 1998; Wolosker et al. Ser and at the same time blocking the glycine transporter 2000) and adrenal glands (Sakai et al. 1997), then in brain Glyt1. This means that d-Ser is the dominant co-agonist and peripheral tissues (Dunlop et al. 1986; Hashimoto for NMDA receptor-elicited neurotoxicity mediating all et al. 1995). The amount of d-Asp in these organs increases cell death elicited by NMDA in organotypic slices. This after birth and reaches its maximum value with tissue result implies an essential role for d-Ser with implication maturation. for the mechanism of neuronal death in the nervous system D’Aniello et al. (1996) found by immunocytochemi- (Shleper et al. 2005). cal techniques intrinsic d-Asp in Leydig and Sertoli cells, There is ongoing debate on where d-Ser is produced, in and also found that intraperitoneal administration of d-Asp neurons or in astrocytes. Recent results from several groups increased the level of d-Asp in the testes and in serum tes- have shown that d-Ser is predominantly made in neurons. tosterone. Other researchers (Wang et al. 2002) showed that Several authors investigate the pathways for d-Ser release intrinsic d-Asp is subcellularly localized to the heterochro- using primary neuronal cultures, and brain slices. Kartvel- matin in the nuclei of magnocellular neurosecretory neu- ishvily et al. (2006) and Rosenberg et al. (2010) found that rons and nucleoli of the cells synthesizing oxytocin in the d-Ser is released by neuronal depolarization both in vitro rat hypothalamus. In addition, Wang et al. (2002) showed and in vivo. Neurotoxin veratridine (a depolarizing agent) that exogenous d-Asp stimulates expression of oxytocin or depolarization in the presence of KCl elicits a signifcant mRNA. release of endogenous d-Ser from primary neuronal cul- To study the regulation of d-Asp, Errico et al. (2006) tures, not from astrocytes. developed mouse strains defcient in d-Asp oxidase. In

1 3 An overview on d-amino acids these defcient mice, d-Asp accumulated in kidney, brain classes of microorganisms. In fact, d-Glu and d-Asp can and spleen to levels 10–25 times higher than normal. The serve as indicators for proteins of bacterial origin (Csapò increased levels of d-Asp signifcantly increased NMDA et al. 2001) and their concentrations are correlated with in the brain. Huang et al. (2006) found that d-Asp accu- the growth of probiotic bacteria during fermentation in mulated in the pituitary while the melanocortin synthesis ewes’ and goats’ milk and whey products (Kehagias et al. decreased in d-Asp oxidase-defcient mice. 2008). Bacteria from oral and intestinal fora, and rumen A high concentration of d-Asp is observed in embryos. It microorganisms are potential source of dietary d-amino disappears in nervous tissues after delivery, but it increases acids (Rooke et al. 1984; Brückner et al. 1992). temporarily in endocrine glands, such as in the pituitary, Some natural and synthetic peptides, which contain pineal and adrenal glands at the specifc stages (Furuchi d-amino acids, have strong antimicrobial properties. Syn- and Homma 2005). In addition, d-Asp levels increase in thetic peptides, which inactivate Clostridium botulinum, testes just before birth and during maturation. Probably this contain glycyl-d-Ala, myristoyl-d-Asp and sorbyl-d-Trp d-amino acid is synthesized by the pituitary gland and by (Paquet and Rayman 1987). In natural antibiotics, d- the testes, where d-Asp, produced inside the seminiferous Asp, d-Glu, d-Phe and d-ornithine (d-Orn) are present in tubules, acts on Leydig cells following testosterone synthe- bacitracin; d-Val in penicillin G; d-AAs in cephalosporin sis enhancement by activating the expression of Steroido- C; d-Ala, d-leucine (d-Leu) and d-Val in actinomycin, genic Acute Regulatory (StAR) protein. Mammalian cells gramicidin and valinomycin; d-Asp and d-Glu in myco- appear to contain all the molecular components required to bacillin; d-Phe and d-Trp in fungisporin, tyrocidine A, B, regulate d-Asp homeostasis, as they can synthesize, release, C, D and pleurocidin (Sarges and Witkop 1965; Jack and take up, and degrade the amino acid. These fndings collec- Jung 1998; Lee and Lee 2008). The mechanism of action tively indicate that d-Asp is a novel type of messenger in of these antibiotic peptides involves the formation of ion the mammalian body (Furuchi and Homma 2005). channel pores spanning the lipid membranes that cause lysis and death of cell with disruption of bacterial membranes (Chattopadhyay and Kelkar 2005) (Table 2). d‑Amino acids in microorganisms, in natural As frequent use of antibiotics is generating resist- and synthetic antibiotics ant strains of bacteria, one of the strategies, followed by the investigators, is to synthetically modify the structure Microbes provide a potential source of d-amino acids, of naturally occurring antibiotics to create new drugs producing, using and metabolizing them. The d-amino (Kalman and Barriere 1990; Rolinson and Geddes 2007). acids produced by several classes of bacteria (Aceto- The most prominent examples are the β-lactam penicil- bacter, Lactobacillus, Micrococcus and Streptococcus), lin and cephalosporin. 7-Aminocephalosporanic acid utilized in starter cultures for the productions of fer- (7-ACA) is a starting compound for the synthesis of vari- mented food and beverages, have been determined in the ous cephalosporin of different generation. Now, 7-ACA Brückner laboratory (1992). In all bacteria, d-Ala and is produced from cephalosporin C with a bio-catalytic d-Asp were found in high concentrations; besides, the method in the presence of Trigonopsis variabilis DAAO dipeptide d-Ala-d-Ala contributes to the antibiotic resist- and glutaryl-7-ACA hydrolase (Pilone and Pollegioni ance (Reynolds 1998). d-Glu is also present in several 2002; Tishkov et al. 2008). Chemical modifcations of

Table 2 d-Aminoacid in bacterial peptides

Drug d-Aminoacid Source Activity

Actinomycins d-Val Bacillus brevis Antibiotic Gramicidin D d-Leu, d-Val Bacillus brevis Antibiotic (permeabilizes lipid membranes by forming ion channels) Gramicidin S d-Phe Bacillus brevis Antibiotic (membrane disruption of lipid bilayer) Bacitracins d-Asp, d-Glu, d-Phe Bacillus brevis Antibiotic Tyrocidines A, B d-Phe, d-Tyr Bacillus brevis Antibiotic (permeabilizes lipid membranes) Monamycins d-Val, d-Ile Streptomyces strains Antibiotic Penicillin G d-Val Penicillium and Aspergillus strains Antibiotic used to treat bacterial infections Cephalosporin C d-AAs Cephalosporium acremonium (fungus) Antibiotic used to treat bacterial infections Vancomycin d-Leu, d-pHPG Amycolatopsis orientalis Antibiotic (inhibits cell wall synthesis in Gram-positive bacteria)

1 3 G. Genchi

6-aminopenicillanic acid (6-APA) and 7-ACA led to might cause pathology. This might yield novel diagnostic the preparation of many clinically used semisynthetic and therapeutic strategies. penicillins and cephalosporins, as amoxicillin (d-Val, A negative effect of the presence of d-amino acids in d-pHPG/d-phydroxyphenylglycine), ampicillin (d-Val, foods is the decrease of proteins’ digestibility and their d-PG/d-phenylglycine), benzylpenicillin and cloxacillin. nutritional value. In some cases low digestibility of proteins

A synthetic all-d-peptide [(RLA)2R]2 is bacteriostatic can be used in weight control diet. Enzymatic hydrolysis against E. coli. This peptide is neither hemolytic nor cyto- of proteins containing d-amino acids at peptide bonds with toxic against mammals thanks to its stability to enzymatic l-amino acids is lower than that of native proteins. c-DNA degradation it may be designated as new potent bacterial cloning has shown that at those positions where d-amino agent (Ryadnov et al. 2002). Another potent new lipopep- acids are found at the end protein, a normal codon for the tide antibiotic, A54145E (Asn3Asp9), containing d-amino corresponding l-amino acid is present (Kreil 1997). This acids, was identifed and isolated from the fermentation means that d-stereoisomers are formed from l-amino acids broth of genetically engineered strain of Streptomyces fra- thanks to posttranslational reactions. diae DA1489 (Gu et al. 2010). KKVVFKVKFKK, a synthetic antimicrobial peptide, Acknowledgements The author gratefully acknowledges the fnan- which acts on the lipid membrane of pathogens, was used cial support provided by Ministero dell’Istruzione, dell’Università e della Ricerca, Italia (MIUR). The author also thanks Dr. Adelaide to investigate the effect of d-amino acid substitution on sta- Romito for English revision. bility, secondary structure and activity. d-Amino acid sub- stitutions at the N- and/or C-terminal of this peptide, which Compliance with ethical standards had little effect on the alpha-helical structure, maintained antimicrobial activity. Instead, the substitution of a d-amino Confict of interest The author reports that there are no conficts of acid in the middle of the amino acids sequence, disrupting interest. the alpha-helical structure, resulted in the complete loss of activity. This result suggests that partial d-amino acid substitution is a useful technique to improve the in vivo activity of antimicrobial peptides (Hong et al. 1999). References

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