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DOI 10.1007/s00726-017-2459-5
Amino Acids
REVIEW ARTICLE
An overview on d‑amino acids
Giuseppe Genchi1
Received: 19 April 2017 / Accepted: 26 June 2017
© Springer-Verlag GmbH Austria 2017
Keywords d-amino acid · Amino acid racemase · d-amino
acid oxidase · d-Asp · d-Ser
Introduction
Proteins are the most abundant biological macromolecules
occurring in all cells and all parts of cells. All proteins, both
from bacteria and from the most complex forms of life, are
composed of 20 amino acids, covalently linked through
a peptide bond in a myriad of different combinations and
sequences. From these 20 building blocks, all organisms can
turn out different products such as enzymes, hormones, trans-
porters, antibodies, lens proteins, haemoglobin transporting
oxygen, cytochromes transporting electrons, antibiotics and a
myriad of other substances with distinct biological activities.
All 20 standard amino acids found in proteins are
α-amino acids, i.e. the general structure of the amino
acids includes a carboxyl group and an amino group, both
bonded to the α-carbon atom (the one next to the carboxyl
group); this α-carbon is also linked both to a hydrogen and
to a side chain group.
There are about 500 amino acids in nature, but only 20
are proteogenic. Proteins are synthetized on polysomes in the
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 poly-
peptide must fold into its proper three-dimensional confor-
mation after posttranslational reactions. The new polypeptide
can undergo enzymatic reaction, including the formation of
disulfide bridges; addition of methyl, carboxyl, acetyl, phos-
phoryl, palmitoyl, retinoyl, or other groups to some amino acid
residues; proteolytic cleavage; attachment of oligosaccharides
or prosthetic groups. Amino terminus may be blocked, and dif-
ferent moieties, small and large, can be added to the reactive
Abstract More than half a century ago researchers thought
that d-amino acids had a minor function compared to
l-enantiomers in biological processes. Many evidences
have shown that d-amino acids are present in high concen-
tration in microorganisms, plants, mammals and humans
and fulfil specific biological functions. In the brain of mam-
mals, d-serine (d-Ser) acts as a co-agonist of the N-methyl-
d-aspartate (NMDA)-type glutamate receptors, responsible
for learning, memory and behaviour. d-Ser metabolism is
relevant for disorders associated with an altered function of
the NMDA receptor, such as schizophrenia, ischemia, epi-
lepsy and neurodegenerative disorders. On the other hand,
d-aspartate (d-Asp) is one of the major regulators of adult
neurogenesis and plays an important role in the develop-
ment of endocrine function. d-Asp is present in the neu-
roendocrine and endocrine tissues and testes, and regulates
the synthesis and secretion of hormones and spermatogen-
esis. Also food proteins contain d-amino acids that are nat-
urally originated or processing-induced under conditions
such as high temperatures, acid and alkali treatments and
fermentation processes. The presence of d-amino acids in
dairy products denotes thermal and alkaline treatments and
microbial contamination. Two enzymes are involved in the
metabolism of d-amino acids: amino acid racemase in the
synthesis and d-amino acid oxidase in the degradation.
Handling Editor: J. D. Wade.
* Giuseppe Genchi
giuseppe.genchi@unical.it
1 Dipartimento di Farmacia e Scienze della Salute e della
Nutrizione, Università della Calabria (UNICAL), Arcavacata
di Rende, Cosenza 87036, Italy
G. Genchi
1 3
groups of proteins with covalent modification reactions. As a
result of carbohydrates addition, biophysical properties as well
as biological activities and stability increase, interaction with
membrane receptors or nucleic acids can change and enzymes
catalytic properties can depend on side chain modification.
Besides, the reversible phosphorylation of serine, threonine
and tyrosine (Burnett and Kennedy 1954) is greatly important
for the entire network of intracellular signalling. A new type
of posttranslational reaction has been revealed about 50 years
ago, i.e. the conversion of certain amino acids in peptides or
proteins from the l- to the d-configuration (Bevins and Zasloff
1990; Kreil 1997).
Stereochemistry of α‑amino acids
Every object has a mirror image and the elements of the pair
of objects, that are mirror images, can be superimposed on
each other. In other cases, the mirror image objects cannot
be superimposed on each other, but are related to each other
as the right hand is to the left hand. Such not-superimpos-
able mirror images are said to be chiral (from the classic
Greek “χειρ”, hand). Frequently a chiral centre in biomole-
cules is a carbon atom (sp3 hybridized) linked to four differ-
ent substituents. For all standard amino acids, the α-carbon
is bonded to four different groups: a carboxyl group (–
COOH), an amine group (–NH2), a hydrogen (–H) and a
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 ste-
reoisomers. Isoleucine and threonine have four stereoi-
somers, 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 contain-
ing 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 excep-
tion of the rotation of the plane of polarized light in dif-
ferent directions, i.e. dextrorotatory or levorotatory.
Special nomenclature has been employed to specify the
absolute configurations of the four substituents of asym-
metric carbon atoms. The absolute configurations of simple
sugar and amino acids are specified by the l- and d-systems
based on the absolute configuration proposed by Emil Fis-
cher (1891) of the three-carbon atom glyceraldehyde, the
smallest sugar to have an asymmetric carbon atom. For
all chiral compounds, stereoisomers with a configuration
related to that of l-glyceraldehyde are designated L, while
stereoisomers related to d-glyceraldehyde are designated
D.
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
and methyl group (–CH3) downwards. The –OH group on
the chiral carbon is left in the l-stereoisomer and right in
the d-stereoisomer (Fig. 1). The amino acid alanine will
be written with the carboxyl group (–COOH) upward, and
the methyl group (–CH3) downward. The –NH2 group on
the chiral carbon is toward the left in the l-stereoisomer
and to the right in the d-stereoisomer (Fig. 1). It is impor-
tant to note that not all l-amino acids are L levorotary
(rotating plane-polarized light to the left), but they can
rotate the plane of polarized light to the right; and in the
same way not all d-amino acids are D dextrorotary (rotat-
ing plane-polarized light to the right), but they can rotate
the plane of polarized light to the left. By E. Fischer’s
Fig. 1 Relationship of stereoisomers of alanine with the absolute
configuration of l- and d-glyceraldehyde
An overview on d-amino acids
1 3
convention, l and d refer only the absolute configuration
of the four substituents around the chiral atom.
d‑Amino acids of dietary origin
Food proteins usually contain l-α-amino acids, but some
d-isomers occur in food either naturally originated or
processing-induced under specific conditions such as
high temperatures, strong acid and alkali treatments,
fermentation processes or cases of non-fermented foods
adulteration (Hayase et al. 1975; Friedman et al. 1984;
Chiavaro et al. 1998).
Food stores prepare and sell increasing quantities of
foods (baked potatoes, fruit juices and fruit pulp, break-
fast cereals, tomato sauces, milk, etc.), which in some
cases contain substantial quantities of d-amino acids
(Csapò et al. 2009). In these foods, the racemization pro-
cess is responsible for the formation of d-amino acids.
The principal factors influencing racemization are alka-
line and acid pHs, treatment duration, heat treatment and
duration of heating. Milk, meat and fruit juices, which do
not contain substantial quantities of d-amino acids, are
often exposed, in the course of preparation for consump-
tion, to conditions which may give rise to racemization.
Milk and dairy products serve as examples of how
the composition of natural substances can change (Bada
1984). Most dairy products, for example, milk, are first
pasteurized (involving heating for 2–3 min at 75–85 °C)
or ultra-pasteurized (involving heating for very short
period of 1–2 s at 135–145 °C). They are subsequently
subjected to homogenization and condensation, until a
particular product, such as milk for commercial con-
sumption, yoghurt and cheese, derived from the various
milk protein fractions, is finally obtained; yoghurt and
cheese are fermented by means of bacteria, and this pro-
cess also constitutes a source of d-amino acids.
The presence of d-amino acids in dairy products can be
used as a biomarker of thermal and alkaline treatments, and
as adulteration or fortification of the products. A concentra-
tion above 4% of d-alanine (d-Ala) in milk can represent
microbial milk contamination (Oancea and Formaggio
2008); in wines and vinegar the presence of d-proline (d-
Pro) is used as an indicator for age dating (Chiavaro et al.
1998); fermented milk products contain high amounts of d-
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
and fermented beverages (wine, beer and vinegar). The
use of bacteria and yeast in fermentation of sourdough,
honey and liquid spices produce variable amounts of free
d-amino acids (Csapò et al. 2006). Heat treatments and
bacterial activity produce high quantity of d-Ala in fruit
juices (Gandolfi 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 in a variety of foods. Gobbetti et al. (1994)
found that the use of lactic acid and yeast in the fermen-
tation of sourdough before baking results in the produc-
tion of free d-Ala and d-Glu in the dough.
In ewes’ and cows’ milk, d-amino acids originated from
enzymatic digestion of peptides and proteins containing
d-amino acids derived from peptidoglycan proteins of
microbial cell walls in the ruminants’ rumen. In fact, milk
from cows, goats and ewes, but not human milk, con-
tains free d-Ala, d-Asp, d-Glu, d-Ser and d-lysine (d-Lys)
(Albert et al. 2007). In addition, these same amino acids
are present in daily consumed ripened cheeses, and the
d-amino acid content varies among cheeses and changes
during cheese production (Pearce et al. 1988).
The amino acids d-Ala, d-Asp, d-arginine (d-Arg) and
d-Glu are present in fruits such as apples, grapes and
oranges, and also in vegetables such as carrots, tomatoes,
cabbages as well as in the corresponding juices (Gandolfi
et al. 1994; Simó et al. 2004). It is unclear whether these
d-amino acids could originate from plant sources, from
microorganisms present in the soil or from heat treat-
ments used to destroy pathogens. The presence of specific
d-amino acids could be used to differentiate juices from
biologically dissimilar fruits and as an indicator for detect-
ing bacterial activity of fruit juices (Friedman 2010).
A delicacy of traditional Chinese cuisine is repre-
sented by pidan. Duck or chicken eggs are immersed
at room temperature for at least 30 days in an alkaline
solution prepared with 4.2% NaOH and 5% NaCl, which
leads to extensive racemization of all ovalbumin l-amino
acids in d-amino acids with the concurrent formation of
lysinoalanine (Chang et al. 1999).
When we consider the taste of foods, d-amino acids have
sweeter taste compared to l-stereoisomers which generally
have bitter flavour. In some cases, the sweetening power of
d-valine (d-Val), d-phenylalanine (d-Phe) and d-tryptophan
(d-Trp) is higher than that of sucrose (Linden and Lorient
1999). Alitame, an artificial dipeptide sweetener contain-
ing l-Asp and d-Ala, is of commercial interest because it
is about 200 times sweeter than sucrose and about 10 times
sweeter than aspartame (Chattopadhyay et al. 2014).
Amino acid racemase
Two enzymes are chiefly involved in synthesis and degra-
dation of d-amino acids: amino acid racemase and d-amino
acid oxidase (DAAO). The enzyme racemase catalyses the
G. Genchi
1 3
conversion between l- and d-amino acids and can be pyri-
doxal 5′-phosphate (PLP) dependent and PLP independent
(Yoshimura and Esak 2003).
Since l-amino acids are the predominant amino acids
found in living organisms proteins, they act as the sub-
strate to generate d-amino acids. l- to d-amino acids
occur by a reaction in the presence of the enzyme race-
mase that changes the stereochemistry of the chiral
α-carbon in amino acids.
Alanine racemase is a PLP-dependent enzyme that
deprotonates and reprotonates on the opposite side the
α-carbon of l-Ala generating d-Ala. The presence of a
lysine residue in the enzyme active site is essential for
the mechanism of action of this enzyme (Watanabe et al.
1999). d-Glu is a component of the peptidoglycan cell
wall in bacteria; d-Glu is produced by a PLP-independent
glutamate racemase (Choi et al. 1992) with two cysteines
involved in the catalysis. In addition, d-Asp occurs in
the peptidoglycan layer of same bacterial cell walls and
is produced from l-Asp in the presence of aspartate
racemase, an enzyme PLP independent as glutamate
racemase. An essential cysteine residue is present in the
enzyme active site (Yamauchi et al. 1992).
In mammals, the enzyme serine racemase (Srr) converts
l- to d-Ser in the presence of PLP (Fig. 2), Mg++ and ATP
(Wolosker et al. 1999; De Miranda et al. 2002). Also Ca++
or Mn++ was necessary for enzyme activity, whereas the
presence of chelators such as ethylenediaminetetraacetic
acid (EDTA) completely inhibited the enzyme serine race-
mase (Cook et al. 2002). By contrast, glycine, l-aspartic
acid and l-asparagine competitively inhibit this enzyme
(Dunlop and Neidle 2005). Serine racemase is also strongly
inhibited by reagents that react with sulfhydryl groups such
as glutathione. In addition, serine racemase converts d- to
l-Ser albeit with lower affinity (Wolosker et al. 1999).
Many aspects regulating d-Ser production under physi-
ological and pathological conditions are to be elucidated.
In the Wolosker lab, Kolodney et al. (2015) investigated the
mechanisms that regulate the synthesis of d-Ser by serine
racemase in paradigms relevant to neurotoxicity. Kolodney
in his paper reports that serine racemase undergoes nucleo-
cytoplasmic shuttling and that this process is dysregulated
L
-serine
A
N
OH
CH
3
C
HO
H
CCOO
-
H
NH
3
+
HOH
2
C
+
N
OH
CH
3
C
HNH
+
H
C COO
-
H
HOH
2
C
Schiff base
H
+
H
+
PP
N
OH
CH
3
C
HN
H
CCOO
-
HOH
2
C
P
++
H
+
N
OH
CH
3
C
HNH
+
H
C COO
-
H
HOH
2
C
P
N
OH
CH
3
C
HO
H
P
+
D
-serine
CCOO
-
H
NH
3
+
HOH
2
C
+
+
N
OH
CH
3
C
HNH
+
H
C COO
-
H
HOH
2
C
P
+
H
2
O
N
OH
CH
3
C
HNH
+
H
CCOO
-
CH
2
P
+
N
OH
CH
3
C
HO
H
P
+
C
NH
3
+
COO
-
CH
2
+
C
O
COO
-
CH
3+
NH
4
+
B
N
OH
CH
3
C
HN
H
CCOO
-
HOH
2
C
P
H
+
H
2
O
Fig. 2 Serine racemase pyridoxal-5′-phosphate dependent. This
enzyme catalyses a the racemization between l-serine and d-serine
and b the α,β-elimination of water from l-serine or d-serine to pro-
duce iminopyruvate, which non-enzymatically hydrolyses to form
pyruvate and ammonia
An overview on d-amino acids
1 3
by several insults leading to neuronal death by apoptotic
stimuli. In this way, cell death induction promotes nuclear
accumulation of serine racemase, nuclear translocation of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
and seven in absentia homolog (Siah) proteins (that func-
tion as E3 ubiquitin ligase in proteins degradation) at the
early stage of cell death process. As a consequence of this
apoptotic insult, nuclear serine racemase and GAPDH are
completely inactivated and this implies that extracellular d-
Ser concentration is drastically reduced, while extracellular
glutamate concentration increases several times.
Human beings can acquire d-amino acids through inges-
tion of food, derivation from endogenous microbial flora,
liberation from metabolically unstable polypeptides, con-
taining d-amino acids after racemization with ageing, and
through biosynthesis from l-amino acids.
Foltyn et al. (2005) and De Miranda et al. (2002) found
that serine racemase catalyses also the α, β-elimination of
water from both l- and d-Ser producing pyruvate and ammo-
nia (Fig. 2). Pyruvate is further metabolized by pyruvate
dehydrogenase complex to acetyl-CoA and by Krebs cycle.
d-Asp is located in the nervous and reproductive systems
with various physiological roles. Whereas several lines of
evidence suggest that this amino acid has an endogenous
origin, the enzyme responsible for mammalian d-Asp
biosynthesis has not yet been identified. Ito et al. (2016)
showed that mammalian enzyme serine racemase, the pri-
mary enzyme responsible for brain d-Ser production also
catalyses Asp racemization. The authors observed that
overexpression of serine racemase in rat pheochromocy-
toma PC12 cells resulted in an increase in intracellular d-
Asp compared with control cells, demonstrating that this
enzyme functions as an Asp racemase (Ito et al. 2016).
d‑Amino acid oxidase (DAAO)
Despite of their beneficial role in some diseases
(for example, d-Ser in schizophrenia, epilepsy and
neurodegenerative disorders; Fuchs et al. 2005), d-amino
acids could have negative effects in diet, first of all affect-
ing the digestibility of food protein and the availability of
the other l-amino acids (Man and Bada 1987). In fact,
the peptide bonds with d-amino acids are more resistant
to proteases. This process results in a reduction of the
quantity of the essential l-amino acids, since the peptide
bonds cannot split in the normal way.
DAAO was first described by Krebs (1935). This
enzyme is widespread in nature from microorganisms to
invertebrates, plants, vertebrates and mammals. The mito-
chondrial enzyme DAAO is an FAD-dependent enzyme
that catalyses the oxidative deamination of d-amino acid,
yielding hydrogen peroxide (H2O2) and an imino acid
(Fig. 3). This latter is non-enzymatically hydrolyzed to
an α-ketoacid and ammonium (NH4+). The α-ketoacids
can undergo a transamination enzymatic reaction PLP
dependent, which results in the l-enantiomer of the origi-
nal amino acid, which in turn enters the usual metabolic
processes, or alternatively is broken down in another
reaction, like oxidative decarboxylation. A characteristic
of all the DAAO is their high specificity towards d-amino
acids, while they are inactive towards the corresponding
l-amino acids. DAAO exhibits optimal activity towards
free neutral d-amino acids, and marginal activity towards
basic ones. Acidic amino acids are oxidized by another
flavo-enzyme, d-aspartate oxidase.
DAAO is used as a bio-catalyst in several biotechno-
logical applications, such as the oxidation of cephalo-
sporin C, and as the biological component in several bio-
sensors for the determination of the content in d-amino
acids of biological fluids. To determine d-Ser levels in
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-
m-phenylenediamine and a layer of immobilized DAAO
from the yeast Rhodotorula gracilis. When implanted
in the cortex of anesthetized rats, this microbiosensor
detected the increase in concentration of d-Ser resulting
H
COOHR
NH
2
DAAO
FADFADH
2
H
2
O
2
O
2
aminoacid
COOHR
NH
iminoacid
OH
2
COOHR
O
+
NH
3
α-ketoacid
Fig. 3 Scheme of the reaction catalysed by the d-amino acid oxi-
dase (DAAO). The d-amino acid is oxidized to an imino acid in the
presence of the enzyme d-amino acid oxidase. FAD (flavin adenine
nucleotide) is reduced to FADH2. In turn, FADH2 is re-oxidized
in the presence of O2 producing FAD and H2O2. The imino acid is
non-enzymatically hydrolyzed to the corresponding α-ketoacid and
ammonia
G. Genchi
1 3
from its diffusion across the blood–brain barrier after an
intraperitoneal injection. This device will make it possi-
ble to investigate in vivo the variations in d-Ser concentra-
tions occurring under normal and pathological conditions.
d‑Amino acids in living organisms
Three different hypotheses have been formulated to explain
the presence of d-amino acids in living organisms’ proteins.
It may result from different mechanisms such as direct
incorporation in the peptide chain of a d-amino acid (pro-
duced for example by amino acid racemase), non-enzy-
matic racemization associated with ageing or diseases, and
enzymatic posttranslational modification. In the last case,
the peptides containing d-amino acids are synthetized via
a ribosome-dependent manner, and a normal codon for
l-amino acid is present in the mRNA at the position where
the d-amino acid is processed in the biologically active
peptide by peptidyl aminoacyl l-d isomerization (Ollivaux
et al. 2014).
Free d-amino acids and peptides containing d-amino
acids have been isolated from a great variety of organisms.
For example, bacterial cell wall peptidoglycans contain d-
Asp, d-asparagine (d-Asn), d-Glu, d-glutamine (d-Gln) and
d-Ala (Reaveley and Burge 1972; Bada et al. 1983; Csapò
and Henics 1991), playing an important role in the bacte-
ria resistance to proteolytic digestion (Tipper and Wright
1979). In Gram-negative bacteria, a single layer of pepti-
doglycans is sufficient to maintain the mechanical stability
of the cell, while in Gram-positive bacteria, which lack an
outer cell membrane, the cell wall is thicker because of sev-
eral layers of peptidoglycans. d-Ala and d-Glu (Cava et al.
2011) are the most common amino acids present in the
bacterial cell wall; however, in the peptidoglycans of some
bacteria are present other d-amino acids such as d-Asp and
d-Ser (Veiga et al. 2006).
In some marine worms and invertebrates, the cellular
fluid contains d-amino acids as a main component (Corri-
gan 1969; D’Aniello and Giuditta 1978; Matsushima et al.
1984; Felbeck 1985); in certain marine shellfish quanti-
ties of d-amino acids can exceed 1% (Preston 1987). Also
higher plants contain d-amino acids (Robinson 1976). d-
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
and lentils (Brückner and Westhauser 1994).
Numerous studies have shown that the secretion of the
skin of amphibians contains a large variety of biologically
active peptides, which have proved to be homologous or
identical to mammalian hormones and/or neurotransmit-
ters. After isolation of enkephalins from the human brain,
Erspamer began to search for similar peptides in the skin
of many amphibians. In the lab of Erspamer, Montecucchi
et al. (1981) isolated from the skin of tree frog Phyllome-
dusa sauvagei the dermorphin, a heptapeptide containing
d-Met, which binds with high affinity to μ-type opioid
receptors (Table 1). Erspamer et al. (1989, 1993) studied
the deltorphin, a linear heptapeptide present on the skin of
tree frog Phyllomedusa bicolor containing d-Ala, which
acts as potent hallucinogen. This peptide shows an high
affinity for δ receptors (Table 1). These peptides have the
sequence Tyr-d-Xaa-Phe-Asp(or Glu)-Val-Val-Gly-NH2,
where d-Xaa is d-Met for dermorphin and d-Ala for deltor-
phin. Research carried by Jimenez et al. (1996) showed that
an octapeptide found in the venom of the fish-hunting snail
Conus radiatus contains a d-Trp residue (Table 1).
In humans, d-amino acids are considered physiologi-
cally active compounds and markers of diseases, deriving
from racemization of l-isomers (Hamase 2007). Wolosker
et al. (1999) have shown that d-Ser plays an important
role in both physiological and pathological processes in
cerebral cortex. In other cases, proteins containing d-Asp
are formed owing to ageing of certain tissues, includ-
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
fish
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
An overview on d-amino acids
1 3
femur) (Ohtani et al. 1998) and eye cataracts (Fujii et al.
1999).
In our life, d-amino acids may have important biologi-
cal effects. First of all, they may be enzymatically con-
verted to l-amino acids by DAAO, and thus they provide
a pool for amino acids necessary for synthesis of proteins
and for anaplerotic reactions of the Krebs cycle (Asp and
Glu). d-Amino acids may act antagonistically to l-amino
acids thanks to deactivation the binding to a biological site
(Friedman and Levin 2012).
d-Amino acids play roles in human physiology and
pathology. The most abundant d-amino acids in mammals
are d-Ser and d-Asp. Fuchs et al. (2005) have studied the
presence of these two amino acids in the central nervous
system. d-Ser has an important role as neuromodulator of
glutamatergic neurotransmission (Miller 2004; Bauer et al.
2005; Fuchs et al. 2005) and is also present in the verte-
brate retina (Estevens et al. 2003). d-Asp has a beneficial
role for reproduction (D’Aniello et al. 2005) and is impli-
cated in neuroendocrine functions (D’Aniello 2007). d-Ser
and d-Ala have been implicated or implied in pathophysiol-
ogy of Alzheimer’s disease (Hamase et al. 2002).
d-Ser and d-Asp are synthesized by serine racemase
and aspartate racemase and are, respectively, degraded by
DAAO and DDO. The presence of d-amino acids in physi-
ological fluids is influenced by age, diet, physiological state
and antibiotic therapies. It is known, in fact, that peptides
containing natural and synthetic d-amino acids are used as
antibiotics. These antibiotics include gramicidin, bacitra-
cin, actinomycin, valinomycin, tyrocidine and many others.
These antibiotics act by disrupting membranes of bacterial
cell through ion channel formation (Chattopadhyay and
Kelkar 2005).
To determine d-amino acids in mammals, sensitive and
selective methods are needed, such as gas chromatogra-
phy, high-performance liquid chromatography, high-per-
formance capillary electrophoresis (Zhao et al. 2001) and
enzymatic activities (Hamase et al. 2002). With regard to
d-amino acid-containing peptides, mass spectrometry is
another option to identify these amino acids (Koehbach
et al. 2016).
Presence and function of d‑Ser in mammals
First in 1992, Hashimoto et al. (1992) found d-Ser in the rat
frontal brain area, representing about 20–25% of the total
amount of serine (d-Ser plus l-Ser). Subsequently, naturally
occurring d-Ser was found not only in the brain (Hashimoto
et al. 1992; Hamase et al. 1997; Hashimoto and Oka 1997),
but also in peripheral tissue (Hashimoto et al. 1992; Hashi-
moto and Oka 1997; Sakai et al. 1998) and physiological
fluids (Hashimoto et al. 1993). Within the brain of various
species of mammals, d-Ser is especially localized to the
frontal brain areas, such as hippocampus, hypothalamus
and striatum (Hashimoto et al. 1995), while the amount of
this d-enantiomer is small in cerebellum, medulla oblongata
and spinal cord (Hashimoto et al. 1995). In neonatal rats and
mouse cerebellum, a high amount of d-Ser is present, which
decreases with age; throughout the life span d-Ser remains
present in the frontal brain areas (Hashimoto et al. 1995).
Studies in vivo and in vitro have suggested that d-amino
acids and especially d-Ser have an important role in N-methyl-
d-aspartate (NMDA) receptor-mediated neurotransmission
(Shleper et al. 2005; Wolosker et al. 2008). The NMDA
receptors are involved in important functions in physiologi-
cal and pathophysiological processes, such as learning and
memory (Collingridge 1987), synaptic formation (Danysz
and Parsons 1998), epilepsy (Giraldez and Girardi 2000) and
nociception (Palazzo et al. 2002). The NMDA receptor has
several modulation sites, where both glycine and d-Ser bind
with high affinity (Schell et al. 1997a). The naturally occur-
ring d-Ser is localized in close vicinity to NMDA receptor,
while glycine is differently distributed from d-Ser and the
NMDA receptors (Hashimoto and Oka 1997; Schell et al.
1997a). d-Ser serves as a co-agonist of the NMDA receptor
in mammalian brains, and its behaviour is probably related
to neurological disorders such as Alzheimer’s disease and
amyotrophic lateral sclerosis. Mothet and co-authors showed
that depletion of d-Ser in the presence of DAAO attenuates
NMDA receptor-mediated neurotransmission, demonstrating
that d-Ser has an important role in binding to glycine site of
the NMDA receptor (Mothet et al. 2000).
A number of evidences suggest that hypofunction of
glutamatergic neurotransmission via the NMDA receptor
plays a crucial role in the pathophysiology of schizophrenia
(Krysta et al. 1999; Hashimoto 2006). d-Ser, synthesized,
as already mentioned, from l-Ser by serine racemase is an
endogenous co-agonist of the NMDA receptor (Wolosker
et al. 1999). It is important to remember that studies with
serine racemase (Srr) knockout mice showed that levels of
d-Ser in the forebrain of Srr knockout mice are 80–90%
lower than in wild-type mice (Horio et al. 2011). This result
explains that d-Ser production in forebrain is dependent on
serine racemase activity.
Several studies have shown that disturbed NMDA recep-
tor neurotransmission, due to decreased d-Ser levels, is a
factor that causes schizophrenia (Wang et al. 2001; Coyle
and Tsai 2004; Hashimoto 2006; Ferraris and Tsukamoto
2011; Labrie et al. 2012). Therefore, treatment with d-Ser
is beneficial to reduce positive, negative and cognitive
symptoms in patients with schizophrenia (Tsai et al. 1998).
As the NMDA receptor is related to some important physi-
ological and pathological processes, it has triggered the
idea of using d-Ser as a therapeutic drug, especially for the
treatment of schizophrenia.
G. Genchi
1 3
But before considering d-Ser a therapeutic agent, it
would be necessary to solve the problem of its bioavail-
ability. When d-Ser is orally administered, DAAO imme-
diately metabolizes this d-amino acid, diminishing its bio-
availability. Therapeutic levels of d-Ser could be achieved
if d-Ser is co-administered with DAAO inhibitors (Ferraris
and Tsukamoto 2011; Sacchi et al. 2013; Hin et al. 2016).
Antipsychotic agents targeting dopamine D2 receptors are
generally used for the treatment of schizophrenia. There-
fore, a new antipsychotic agent has to be proposed as a new
strategy. In fact, d-Ser administration to laboratory rodents
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
tissues. d-Ser is a physiological endogenous co-agonist at
the glycine site of N-methyl-d-aspartate (NMDA) subtype
of glutamate receptors. d-Ser is synthesized by pyridoxal-
5′-phosphate-dependent serine racemase. d-Ser is also
essential for neurotransmission, synaptic plasticity and
behaviour. d-Ser may also trigger NMDA-mediated neu-
rotoxicity and its deregulation may play a role in neurode-
generation. d-Ser action has not been previously compared
with that of endogenous glycine, and the relative impor-
tance of the two agonists remains unclear.
Shleper et al. (2005) investigated the efficiency of these
two agonists in mediating NMDA receptors’ neurotoxic-
ity in hippocampal slices. The authors report that removal
of endogenous d-Ser from slices by pretreating the tis-
sue with the enzyme d-Ser deaminase virtually abolished
NMDA-elicited neurotoxicity, but did not protect against
kainate. Although endogenous glycine was ten times more
concentrated than d-Ser, glycine is ineffective in mediating
NMDA receptor neurotoxicity. The effect of endogenous
glycine could be observed by removing endogenous d-
Ser and at the same time blocking the glycine transporter
Glyt1. This means that d-Ser is the dominant co-agonist
for NMDA receptor-elicited neurotoxicity mediating all
cell death elicited by NMDA in organotypic slices. This
result implies an essential role for d-Ser with implication
for the mechanism of neuronal death in the nervous system
(Shleper et al. 2005).
There is ongoing debate on where d-Ser is produced, in
neurons or in astrocytes. Recent results from several groups
have shown that d-Ser is predominantly made in neurons.
Several authors investigate the pathways for d-Ser release
using primary neuronal cultures, and brain slices. Kartvel-
ishvily et al. (2006) and Rosenberg et al. (2010) found that
d-Ser is released by neuronal depolarization both in vitro
and in vivo. Neurotoxin veratridine (a depolarizing agent)
or depolarization in the presence of KCl elicits a significant
release of endogenous d-Ser from primary neuronal cul-
tures, not from astrocytes.
d-Ser is an endogenous ligand for NMDAR gener-
ated from l-Ser by the enzyme serine racemase. Neuronal
and glial localizations have been reported for both d-Ser
and serine racemase. GAPDH is an exclusively astrocytic
enzyme that catalyses the first committed step of l-Ser
biosynthesis. Ehmsen et al. (2013), using transgenic mice
expressing enhanced green fluorescent protein under the
serine racemase promoter and mice with targeted deletion
of serine racemase or GAPDH, demonstrate predominantly
the neuronal sources of d-Ser dependent on astrocytic sup-
ply of l-Ser (Ehmsen et al. 2013).
Presence and function of d‑Asp in mammals
d-Asp is an endogenous amino acid present in invertebrates
and vertebrates and plays an important role in nervous and
neuroendocrine system, as well as in the development of
the nervous system. d-Asp also acts as a neurotransmit-
ter/neuromodulator; indeed, this d-amino acid has been
detected in synaptosomes and in synaptic vesicles, where
it is released after chemical (K+ ion, ionomycin) or elec-
tric stimuli. In the endocrine system, d-Asp is involved in
the regulation of hormone synthesis and release. In the rat
hypothalamus, it enhances gonadotropin-releasing hor-
mone’s (GnRH) release and induces oxytocin and vaso-
pressin mRNA synthesis. In the pituitary gland (D’Aniello
2007), d-Asp stimulates the secretion of the prolactin
(PRL), luteinizing hormone (LH) and growth hormone
(GH). In addition in the testes, it is present in Leydig cells
and is involved in testosterone and progesterone release.
In neonatal and adult rats, d-Asp is predominantly local-
ized in endocrine glands, such as testes (D’Aniello et al.
1996; Schell et al. 1997b; Sakai et al. 1998; Wolosker et al.
2000) and adrenal glands (Sakai et al. 1997), then in brain
and peripheral tissues (Dunlop et al. 1986; Hashimoto
et al. 1995). The amount of d-Asp in these organs increases
after birth and reaches its maximum value with tissue
maturation.
D’Aniello et al. (1996) found by immunocytochemi-
cal techniques intrinsic d-Asp in Leydig and Sertoli cells,
and also found that intraperitoneal administration of d-Asp
increased the level of d-Asp in the testes and in serum tes-
tosterone. Other researchers (Wang et al. 2002) showed that
intrinsic d-Asp is subcellularly localized to the heterochro-
matin in the nuclei of magnocellular neurosecretory neu-
rons and nucleoli of the cells synthesizing oxytocin in the
rat hypothalamus. In addition, Wang et al. (2002) showed
that exogenous d-Asp stimulates expression of oxytocin
mRNA.
To study the regulation of d-Asp, Errico et al. (2006)
developed mouse strains deficient in d-Asp oxidase. In
An overview on d-amino acids
1 3
these deficient mice, d-Asp accumulated in kidney, brain
and spleen to levels 10–25 times higher than normal. The
increased levels of d-Asp significantly increased NMDA
in the brain. Huang et al. (2006) found that d-Asp accu-
mulated in the pituitary while the melanocortin synthesis
decreased in d-Asp oxidase-deficient mice.
A high concentration of d-Asp is observed in embryos. It
disappears in nervous tissues after delivery, but it increases
temporarily in endocrine glands, such as in the pituitary,
pineal and adrenal glands at the specific stages (Furuchi
and Homma 2005). In addition, d-Asp levels increase in
testes just before birth and during maturation. Probably this
d-amino acid is synthesized by the pituitary gland and by
the testes, where d-Asp, produced inside the seminiferous
tubules, acts on Leydig cells following testosterone synthe-
sis enhancement by activating the expression of Steroido-
genic Acute Regulatory (StAR) protein. Mammalian cells
appear to contain all the molecular components required to
regulate d-Asp homeostasis, as they can synthesize, release,
take up, and degrade the amino acid. These findings collec-
tively indicate that d-Asp is a novel type of messenger in
the mammalian body (Furuchi and Homma 2005).
d‑Amino acids in microorganisms, in natural
and synthetic antibiotics
Microbes provide a potential source of d-amino acids,
producing, using and metabolizing them. The d-amino
acids produced by several classes of bacteria (Aceto-
bacter, Lactobacillus, Micrococcus and Streptococcus),
utilized in starter cultures for the productions of fer-
mented food and beverages, have been determined in the
Brückner laboratory (1992). In all bacteria, d-Ala and
d-Asp were found in high concentrations; besides, the
dipeptide d-Ala-d-Ala contributes to the antibiotic resist-
ance (Reynolds 1998). d-Glu is also present in several
classes of microorganisms. In fact, d-Glu and d-Asp can
serve as indicators for proteins of bacterial origin (Csapò
et al. 2001) and their concentrations are correlated with
the growth of probiotic bacteria during fermentation in
ewes’ and goats’ milk and whey products (Kehagias et al.
2008). Bacteria from oral and intestinal flora, and rumen
microorganisms are potential source of dietary d-amino
acids (Rooke et al. 1984; Brückner et al. 1992).
Some natural and synthetic peptides, which contain
d-amino acids, have strong antimicrobial properties. Syn-
thetic peptides, which inactivate Clostridium botulinum,
contain glycyl-d-Ala, myristoyl-d-Asp and sorbyl-d-Trp
(Paquet and Rayman 1987). In natural antibiotics, d-
Asp, d-Glu, d-Phe and d-ornithine (d-Orn) are present in
bacitracin; d-Val in penicillin G; d-AAs in cephalosporin
C; d-Ala, d-leucine (d-Leu) and d-Val in actinomycin,
gramicidin and valinomycin; d-Asp and d-Glu in myco-
bacillin; d-Phe and d-Trp in fungisporin, tyrocidine A, B,
C, D and pleurocidin (Sarges and Witkop 1965; Jack and
Jung 1998; Lee and Lee 2008). The mechanism of action
of these antibiotic peptides involves the formation of ion
channel pores spanning the lipid membranes that cause
lysis and death of cell with disruption of bacterial mem-
branes (Chattopadhyay and Kelkar 2005) (Table 2).
As frequent use of antibiotics is generating resist-
ant strains of bacteria, one of the strategies, followed by
the investigators, is to synthetically modify the structure
of naturally occurring antibiotics to create new drugs
(Kalman and Barriere 1990; Rolinson and Geddes 2007).
The most prominent examples are the β-lactam penicil-
lin and cephalosporin. 7-Aminocephalosporanic acid
(7-ACA) is a starting compound for the synthesis of vari-
ous cephalosporin of different generation. Now, 7-ACA
is produced from cephalosporin C with a bio-catalytic
method in the presence of Trigonopsis variabilis DAAO
and glutaryl-7-ACA hydrolase (Pilone and Pollegioni
2002; Tishkov et al. 2008). Chemical modifications 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
Penicillin G
d-Val, d-Ile
d-Val
Streptomyces strains
Penicillium and Aspergillus strains
Antibiotic
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)
G. Genchi
1 3
6-aminopenicillanic acid (6-APA) and 7-ACA led to
the preparation of many clinically used semisynthetic
penicillins and cephalosporins, as amoxicillin (d-Val,
d-pHPG/d-phydroxyphenylglycine), ampicillin (d-Val,
d-PG/d-phenylglycine), benzylpenicillin and cloxacillin.
A synthetic all-d-peptide [(RLA)2R]2 is bacteriostatic
against E. coli. This peptide is neither hemolytic nor cyto-
toxic against mammals thanks to its stability to enzymatic
degradation it may be designated as new potent bacterial
agent (Ryadnov et al. 2002). Another potent new lipopep-
tide antibiotic, A54145E (Asn3Asp9), containing d-amino
acids, was identified and isolated from the fermentation
broth of genetically engineered strain of Streptomyces fra-
diae DA1489 (Gu et al. 2010).
KKVVFKVKFKK, a synthetic antimicrobial peptide,
which acts on the lipid membrane of pathogens, was used
to investigate the effect of d-amino acid substitution on sta-
bility, secondary structure and activity. d-Amino acid sub-
stitutions at the N- and/or C-terminal of this peptide, which
had little effect on the alpha-helical structure, maintained
antimicrobial activity. Instead, the substitution of a d-amino
acid in the middle of the amino acids sequence, disrupting
the alpha-helical structure, resulted in the complete loss of
activity. This result suggests that partial d-amino acid sub-
stitution is a useful technique to improve the in vivo activ-
ity of antimicrobial peptides (Hong et al. 1999).
Conclusions
In the past it was assumed that only l-amino acids were nat-
urally selected during evolution to synthesize proteins. Up
to 50–60 years ago, it was generally believed that d-amino
acids did not occur in living organisms with the exception
of microorganisms and some peptides. The studies from
the last few decades have without a doubt established the
presence of d-amino acids in higher organisms, including
human beings. This knowledge about d-amino acids is due
to the use of the latest analytical equipment that made it
possible to highlight substantial amount of d-amino acids
in mammals including human beings.
Our knowledge regarding the presence of d-amino
acids in higher species and their interaction with the func-
tion of the brain has markedly increased during the last
few decades. The specific functions of d-amino acids are
far from being unravelled, despite growing understanding
provided by extensive works on d-Ser and d-Asp. d-Ser is
an important neuromodulator and d-Asp has been impli-
cated in developmental and endocrine functions. Moreover,
d-amino acids might also play a mechanistic role in pathol-
ogy. These lines of evidence suggest that d-amino acids are
essential for mammals and that alteration of their amounts
might cause pathology. This might yield novel diagnostic
and therapeutic strategies.
A negative effect of the presence of d-amino acids in
foods is the decrease of proteins’ digestibility and their
nutritional value. In some cases low digestibility of proteins
can be used in weight control diet. Enzymatic hydrolysis
of proteins containing d-amino acids at peptide bonds with
l-amino acids is lower than that of native proteins. c-DNA
cloning has shown that at those positions where d-amino
acids are found at the end protein, a normal codon for the
corresponding l-amino acid is present (Kreil 1997). This
means that d-stereoisomers are formed from l-amino acids
thanks to posttranslational reactions.
Acknowledgements The author gratefully acknowledges the finan-
cial support provided by Ministero dell’Istruzione, dell’Università
e della Ricerca, Italia (MIUR). The author also thanks Dr. Adelaide
Romito for English revision.
Compliance with ethical standards
Conflict of interest The author reports that there are no conflicts of
interest.
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