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“D-amino acids: description and significance” - Loredano Pollegioni


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Watching at the "D" side: D-amino acids and their significance in neurobiology
June 05 -June 09, 2016 – Lake Como School of Advanced Studies

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“D-amino acids: description and significance” - Loredano Pollegioni

  1. 1. “D-amino acids: description and significance” Loredano Pollegioni Università degli studi dell’Insubria 1
  2. 2. W H A T D O N ’ T W E K N O W ? Science, July 20052 A issue of Science journal of july 2005 reported 100 questions that span the sciences (Sciences, 2005, vol. 309). It is expected that some will drive scientific inquire for a long time while others may soon be answered. A very intriguing question was: “what is the origin of homochirality in Nature?” The origin of the preference between mirror-images of natural molecules (such as amino acids and sugars) still remains a mistery.
  3. 3. INTRO on D-AA3 What are D-amino acids? Amino acids are individual building blocks that comprise proteins; they exist in two orientations in the space: levorotatory (L) and dextrorotatory (D), also indicated as left-handed and right-handed. The majority of amino acids in living organisms are L-isomers. In the last few decades with the advancement of technology, the presence of D-AAs was discovered in living things. This finding opened the questions related to their origin, synthesis, and physiological roles. About 500 amino acids are known and they have been classified in many ways: based on the location of the functional groups (α, β, γ, etc. depending on the position of the amino group vs. the carboxylic one), or on the side chain group type, or the isomerism, to be proteinogenic or not, to be of natural origin or not.
  4. 4. 4 All α-amino acids but glycine exist in either of the two enantiomers, which are mirror images of each other: D-AAs do not bind tRNA molecules (Russell Doolittle)*, thus their presence in proteins is related to posttranslational modifications. INTRO on D-AA The left and right handed forms have identical free energy: for the reaction of changing left-handed to right-handed amino acid (L  R), or the reverse, ΔG = 0, so K = 1 (or close)
  5. 5. 5 The importance of chirality Few examples to underline the importance of chirality: 1. Left-handed vs. right handed α-helix 2. Thalidomide: in the 1960s, this drug (produced as a racemate) was prescribed to pregnant women suffering morning sickness. The R- enantiomer of this molecule is a powerful tranqullliser while the S-form can disrupt fetal development resulting in severe birth defects 3. DOPA (3,4-dihydroxyphenylalanine): L-DOPA is effective in the treatment of Parkinson's disease and dopamine-responsive dystonia by stimulating the production of dopamine in the brain, whilst D-DOPA is inactive. 4. L-Glucose is not recognized by red cells transporters 5. Nucleic acid contain D-deoxyribose or D-ribose, only
  6. 6.  Chirality from the greek word for hand. Introduced by Lord Kelvin (1904, Glasgow)  Chirality, an intrinsic geometric property, and stereoisomerism/configuration are distinct and should not be combined  The two forms are non-superimposable mirror images of each other (definition from Kurt Mislow, 1999)  The two forms are called enantiomers or optical isomers because they rotate plane-polarized light either to the right or to the left  The L- and D- convention refers to the optical activity of the isomer of glyceraldehyde (D-glyceraldehyde is dextrorotatory and L-glyceraldehyde is levorotatory) from which the amino acid can be synthesized  The absolute stereochemistry is indicated by the (S) and (R) designators (Cahn-Ingold-Prelog system recommended by IUPAC)  A mixture containing 50:50 of L- and R-enantiomers is called a racemate or a racemic mixture Chirality6
  7. 7. The long story of this terminology: Jons Jakob Berzelius (1779-1884): «isomer» and «isomerism» Aleksandr Butlerov (1828-1886): «chemical structure» Jacobus Henricus van’t Hoff (1874): «asymmetric carbon atom» Aemilius Wunderlich (1886): «configuration» Viktor Meyer (1884-1894): «stereochemistry» William Thompson (1894): «chiral» and «chirality» Louis Pasteur (1848): «dissymetric and dissymetry» (even if he was not the first to use these terms!) Kurt Mislow (1962): publicly introduced «chirality» term into a chemistry paper (JACS) 7 Chirality
  8. 8.  At the early of 1800, the phenomenon of natural optical activity was first observed by the French scientists Arago and Biot  Shortly after, Fresnel developed a theory of optical rotation based on the differential refraction of right- and left-circularized polarized light; the enantiomers show different adsorbtion of circularly polarized UV light Chirality8 - Louis Pasteur in 1848 was the first person on history to resolve a racemate: he used tweezers to separate the left and right-handed crystals of sodium ammonium tartrate (PTA) - Pasteur was fortunate: only two of the 19 chiral amino acids self- resolve in crystalline form and only below 23 °C - The sodium ammonium salt of PTA crystallyzed as a mechanical mixture of two distinct crystal types, they showed optical activity equal in magnitude but opposte in direction
  9. 9. The handedness is complete mistery to evolutionists (is homochirality in the monomeric building-blocks of biopolymers a condition for the initiation of a prebiotic chemistry?) We eat optically active bread & meat, live in houses, wear clothes, and read books made of optically active cellulose. The proteins that make up our muscles, the glycogen in our liver and blood, the enzymes and hormones, the sugars in DNA and RNA and in the metabolic pathways … are all optically active. Naturally occurring substances are optically active because the enzymes which bring about their formation … are optically active. As to the origin of the optically active enzymes, we can only speculate. Origin of Life9
  10. 10. 10 A possibility for origin of homochirality is that it was achieved prior to the origin of life (prebiotic chemistry) In the primordial soup, D- and L-AAs would likely have been present in nearly equal amounts. The preference for L-AAs has been explained differently: a) certain chemical processes were favored energetically leading to increased levels of L-isomers of amino acids on the early earth: the LOH theory*; b) meteorites brought L-AAs to earth thus providing an imbalance that may have favored the formation of life. The AA detected in the Murchison and Murray meteorites have been found to be nonracemic (SETH, search fo extraterrestrial homochirality); c) it is related to the creationistic theory of origin of life. The mystery has not been solved yet! Origin of Life
  11. 11. 11 *According to the LOH-theory (hydrate hypothesis of living matter origination), AAs originated within the methane- hydrate localizations from natural gas, niter, and included sulfur-containing substances at temperatures close to those of living organisms. The geometric differences of the L- and D-forms are rather significant: accordingly, the mechanisms of the sets of elementary steps that lead to their formation are rather different. At 270-290 K the reaction proceeded slowly: the direction of the synthesis and the final optical structure of any amino acid was dictated not by thermodynamic but by kinetic! Racemization is expected but proceeds extremely slow. The primary AAs (and proteins) were produced from natural gas and niter at a low temperature and within solid or semiliquid gas-hydrate structures: under these conditions, L- AAs only were produced that could not transform into the D-form because of the kinetic nature. Origin of Life
  12. 12. 12 Each AA was formed within some cavity and was not in touch with other AA molecules: L-AA is produced that can not be transformed into the D-form. The reactions of formation of L- and D-isomers of AAs proceed with different rates under the same ambient conditions. Racemization is thermodinamically expected but kinetically inhibited as a result of differences in the rates of formation of different optical isomers as a result of the complexity of the intramolecular rearrangements (racemization of asparagine acid ester has a rate of 0.1% per year!) Origin of Life
  13. 13. 13 Origin of homochirality 1. Spontaneous symmetry breaking: a) Spontaneous separations of a racemic mixture into its constituents enantiomers during crystallization (difficult on the primitive Earth) b) Selective adsorption of one enantiomer from a racemic mixture at the surface of a chiral crystal (i.e. quartz crystals) 2. Homochirality arose from statistical fluctuation from the equimolar condition Monod (1970): homochirality of terrestrial life was a necessity and not a matter of chance selection The energy difference between enantiomers is called the parity- violating energy difference (PVED, 10-14 J/mol, corresponding to e.e. of 10-15%). The violation of parity constitutes a symmetry breaking at the level of the basic laws of atomic physics Origin of Chirality
  14. 14. 14 Polymerization of a Glu derivative occurs 20-fold faster starting with molecules of the same chirality (L-L or D-D reactions) rather than with a racemic mixture (L-D or D-L reactions) Biopolymers result from the polymerization of chiral monomers HIV-1 protease was synthesized using D-AAs: it exhibits catalytic activity identical to that of the native enzyme except that it is specific for the opposite enantiomer of the substrate Blackmond group: compounds can strongly influence the e.e. in solution under solid-liquid equilibrium conditions (e.g., valine e.e. increase from 47% to 99% in the presence of fumaric acid) Origin of Chirality
  15. 15. 15 Chemical rules Chiral catalysis: A universal chemical rule states: «Synthesis of chiral compounds from achiral reagents always yields the racemic modification» and «optically inactive reagents yield optically inactive products». To resolve a racemate another homochiral compound is required: actually, (R) and (S) compounds have identical properties except when they interact with other chiral phenomena. E.g., the products of the reaction of the (R) and (S) enantiomers with an (S’) substance generates R-S’ and S-S’ compounds (named diasteroisomers) which are not mirror images and thus differ in physical properties such as solubility. Asymmetric autocatalysis (Franck 1953): the amplification of an e.e. by means of a chemical reaction in which the product is a chiral compound and one enantiomer catalyzes its own formation while inhibiting the formation of the opposite enantiomer (e.g. Soai reaction, 1995)
  16. 16. Peptidoglycan: D-Ala and D-Glu are found in the peptidoglycan cell wall and may contribute to antibiotic resistance in some bacteria D-AAs play a role in bacterial cell wall formation during stationary phase. Many diverse bacterial phyla synthesize and release D-AAs, including D-Met and D-Leu. These D-AAs regulate cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities The primary mode of variation between PG structures is in the length and composition of the interpeptide bridge that can contain: D-Ala, D-Glu, D-Ser, D-Asp, D-Asn, D-Lys and D-Orn. D-Amino Acids and Living Organisms Bacteria: The major sources of D-AA in bacteria are extracellular or periplasmic biomolecules: -- peptidoglycan (the major component of the bacterial cell wall) -- teichoic acids (containing D-AA) -poly-γ-glutamate (containing D- and/or L-Glu, the starting material for “natto”) D-amino acids in bacteria16
  17. 17.  D-AAs in cell culture supernatant impact PG functionality through their incorporation in PG: paracrine regulators in microbial communities. The bacteria release and use D-AAs to decrease cell wall formation when resources are scarce. Thus, the D-AAs help bacteria adapt to adverse environmental conditions.  Periplasmic or extracellular transpeptidases are responsible for incorporation of D-AAs into mature PGs  D-AAs are proposed to disassemble biofilms, to inhibit spore germination and to be produced depending on stress response- related sigma factor RpS (in V. cholera) D-amino acids in bacteria  D-AAs are common constituents of peptide antibiotics synthesized by bacterial non-ribosomal peptide synthetases (NRP)  D-AAs incorporation appears to be largely the domain of prokaryotic organisms  Microbes are likely the major source of D-amino acids in fermented foods (e.g., bread, cheese)
  18. 18. D-Amino Acids and Living Organisms Marine organisms  Marine invertebrates (e.g., crab, shrimp, lobster) may use D-AAs for osmoregulation and/or a source of L-AAs under adverse conditions  D-Asp was found in all microalgae, but D-Ala was only present in the marine diatoms. Venom  The platypus, funnel web spider, and cone snail all have D-AAs in their venom  Isomerases in these organisms are thought to produce the D-AAs needed for the venom. Plants  0,2-8% relative to L-AAs. D-amino acids in living organisms 18
  19. 19. D-Amino Acids and Living Organisms Humans The source of D-AAs for humans is mainly diet Food processing such as high heat converts L-AAs to D-AAs. D-AAs naturally occur in fresh produce Fermented foods (e.g., bread, cheese, vinegar) are high in D-AAs mainly due to microbial activity D-AAs are absorbed by the intestine and travel to tissues where they are metabolized In health: - D-AAs may be important in the brain for neurotransmission and in tumor inhibition - Schizophrenic patients showed increased cognitive function and performance when given a specific D-AA. -D-AAs may also have analgesic (pain reduction) effects D-amino acids in humans19
  20. 20. D-Amino Acids and Living Organisms Among the few D-AAs found in the mammalian central nervous system, D-aspartate together with D-serine (and D-Alanine) are the most abundant (Hashimoto and Oka, 1997) 1986: Dunlop reported the presence of D-AAs in mammals 1992: Hashimoto found large quantities of D-Ser in rat brain 1995: Snyder’s group identified abundant D-Ser in the cerebral cortex, hyppocampus, anterior olfactory nucleus, olfactory tubercle and the amygdala of rats D-amino acids in humans20
  21. 21. Mechanisms for synthesis of D-AAs a – Inversion of -C of the corresponding L-AA (racemases or epimerases) a1. PLP-dependent enzymes* Biochemistry of D-amino acids Mechanism for D-AA synthesis by a PLP- dependent racemase
  22. 22. 22 Biochemistry of D-amino acids a2. cofactor independent enzymes a3. multi-step processes N-acyl-amino acid racemase + N-acyl-D-AA-deacylase b – stereospecific amination of the corresponding -keto acid (D-AA- amino transferases) The substrate specificity of the second half-reaction determines the D-AA formed
  23. 23. D-AAs, responsible enzymes and role Biochemistry of D-amino acids
  24. 24. Biochemistry of D-amino acids D-AAs, responsible enzymes and role
  25. 25. D-serine D-Serine, an atypical signaling molecule, is synthesized from L-serine by the PLP-dependent enzyme serine racemase (EC, SR) D-serine25
  26. 26. D-serine D-Serine is degraded by the PLP-dependent enzyme SR: α,β- elimination reaction D-serine26
  27. 27. D-serine D-Serine is degraded by the FAD-dependent enzyme D-amino acid oxidase (EC, DAAO) D-serine27
  28. 28. Serine shuttle (Wolosker, 2012) D-serine - D-Serine binds to the ‘‘strychnine-insensitive glycine modulatory site’’ of the N-methyl- D-aspartate type glutamate receptor (NMDAR) - It is critically involved in brain development, neuronal cell migration, and plasticity as well as in learning, memory, and excitotoxicity 28
  29. 29. 29 D-serine Levels of D-Ser and D-Ala in different brain regions (Yamanaka et al., 2012) Open bars: Wistar rats Gray bars: SD rats Closed bars: LEA/SENDAI rats (lacking DAAO)
  30. 30. D-aspartate 1986: Dunlop et al. reported that D-asp is present in mammals 1998: Wolosker, Mothet and Snyder identified a mammalian aspartate racemase (DR, EC, which converts L-Asp to D-Asp 2010: Kim et al. reported that DR colocalizes with D-Asp in the brain and neuroendocrine tissues - D-Asp is degraded by the FAD-dependent D-aspartate oxidase (DDO, also abbreviated DASPO; EC Mechanism: - In rat kidney and liver DDO level is induced by exogenous D-Asp D-aspartate30
  31. 31. - D-Asp in the mammalian brain has been mainly focused on its ability to bind and stimulate NMDARs through its direct binding at the glutamate site of this receptor (as agonist) - Moreover, D-Asp may also indirectly stimulate NMDARs because it represents the substrate for the biosynthesis of endogenous NMDA - Very high levels of D-Asp occur transiently during the last stage of the embryonic life or in the post-natal life, suggesting a role in the development of the nervous system (and in neurotransmission) - In thyroid glands production of hydrogen peroxide occurs by oxidation of endogenous D-Asp (by DASPO), required for the synthesis of thyroid hormones - D-Asp as regulator of gene transcription (?) - D-Ala is also present in mammals at moderate levels. Is it synthesized in the body? D-aspartate31
  32. 32. HRP luminol 32 Enzymatic assays DAAO: D-AA + O2  -keto acid + NH3 + + H2O2 HPLC analysis (derivatization with OPA or 1- fluoro.2,4-dinitrophenyl-5-L- alanine amide) light emission Absorbance at 440 (505) nm HRP o-phenylenediamine 10-acetyl-3,7- dihydroxyphenoxazine Fluorescence at 580 nm Oxymeter Hydrazone absorbs at 445 nm 2,4-DNP D-Phegly + O2  benzoyl formic acid (absorbance at 243-252 nm) HRP oDNS (4-AAP) HPLC analysis (derivatization with OPA or 1- fluoro.2,4-dinitrophenyl-5-L- alanine amide) HRP luminol DDO: D-Asp + O2  oxalacetate + NH3 + + H2O2 HRP o-phenylenediamine 10-acetyl-3,7- dihydroxyphenoxazine Fluorescence at 580 nm Oxymeter Hydrazone absorbance at 445 nm 2,4-DNP Absorbance at 440 (505) nm HRP oDNS (4-AAP) light emission
  33. 33. HRP luminol 33 Enzymatic assays SR: L-Ser  D-Ser  OH-pyruvate + NH3 + + H2O2 HPLC analysis (derivatization with OPA or 1-fluoro.2,4- dinitrophenyl-5-L-alanine amide) DAAO HRP o-phenylenediamine 10-acetyl-3,7- dihydroxyphenoxazine Fluorescence at 580 nm Absorbance at 440 (505) nm HRP oDNS (4-AAP) light emission HRP luminol DR: L-Asp  D-Asp  oxalacetate + NH3 + + H2O2 HPLC analysis (derivatization with N-tertOPA or 1- fluoro.2,4-dinitrophenyl-5-L-alanine amide) DDO HRP o-phenylenediamine 10-acetyl-3,7- dihydroxyphenoxazine Fluorescence at 580 nm Absorbance at 440 (505) nm HRP oDNS (4-AAP) light emission
  34. 34. D-amino acids and human pathologies D-serine is involved in:  Schizophrenia: hypofunction of NMDAR because of D-Ser decrease;  familial ALS (fALS): R199W DAAO variant is responsible on motoneurons death induced by excitotoxicity;  addiction: decrease in D-Ser level in nucleo accumbens of rat in abstinence following cocaine treatment;  Alzheimer disease(AD): β-amiloid induces SR expression and D-Ser release;  neuropathic pain: inhibition of DAAO (and of hydrogen peroxide production) gives and analgesic effect in neuropatic pain and bone cancer. D-aspartate is involved in:  increase of testosterone level in male infertility;  brain hormone’s release;  schizophrenia by NMDAR modulation: protective role against sensorimotor- gating deficits;  Alzheimer disease (AD): decreased level in human brains from patients;  Age-related synaptic plasticity deterioration (in mice). 34 Human pathologies
  35. 35. D-AA in biotechnology  Enzymes involved in D-AA synthesis used in PG are key targets for antibacterials  D-AAs are modulators of bacterial growth and persistance (and disassemble mature biofilms)  D-AAs are components of bioactive natural products with different roles: antibacterial, antitumor, antiangiogenic, HIV entry inhibitors, atherosclerosis reduction, βA fibrillogenesis inhibition (in AD)  D-AAs are used in synthetic peptides since they are not recognized by proteases 35 Biotec & D-amino acids
  36. 36. 36 Have a nice (D)-school