A comparison of enzymatic phosphorylation and phosphatidylation of β-l- and β-d-nucleosides

Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates. Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers.     

Ornithine dissimilation byTreponema denticola

Treponema denticola convertedl-ornithine, a product ofl-arginine catabolism, to putrescine via a decarboxylation reaction and to proline via a deamination reaction. Ornithine decarboxylation byT. denticola extracts was stimulated by pyridoxal 5′-phosphate. In the absence of pyridoxal 5′-phosphate, (NH₄)₂SO₄-fractionated extracts converted ornithine to proline and ammonia. This activity was not stimulated by α-keto acids, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide or ADP. Neither ornithine δ-transaminase (l-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) nor Δ¹ reductase [l-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2.] activity was detectable in cell extracts. These results indicate that formation of proline from ornithine inT. denticola is catalyzed by an enzyme system analogous to the ornithine cyclase (deaminating) ofClostridium sporogenes. Exogenous ornithine inhibited the growth ofT. denticola. Thus, in addition to generating putrescine and proline, the ornithine dissimilatory pathways may serve to prevent accumulation of inhibitory concentrations of ornithine in the spirochete's environment.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Pyranose 2-dehydrogenase, a novel sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus

A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (λ_max 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O₂ to H₂O₂ (> 5 × 10⁴-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of ∼79 kDa as determined by gel filtration; its subunit molecular mass is ∼75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag⁺, Hg²⁺, Cu²⁺, and CN^– (10 mM) were inhibitory, while 50 mM acetate had an activating effect. Received: 19 August 1996 / Accepted: 21 November 1996     

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its α,γ-elimination

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH₃SeH from seleno-l-methionine (l-SeMet) by the α,γ-elimination reaction. The l-SeMet α,γ-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The K _m value of the enzyme for the catalysis of l-SeMet was 15.5 m M, and the V _max was 0.29 units/mg protein. Pyridoxal 5′-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50°C; it catalyzed the α,γ-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the α,β-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inerts. Therefore, the purified enzyme was different from the bacterial l-methionine γ-lyase that metabolizes l-SeMet to CH₃SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the α,γ-elimination reaction of l-SeMet and immediately converts it to CH₃SeH, an important metabolite of Se.     

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.     

Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N _ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical retrosynthesis of 2′-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase

2′-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2′-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2′-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2′-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker’s yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5′-triphosphate generated from adenosine 5′-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via d-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2′-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2′-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2′-deoxyribonucleoside synthesis. 2′-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.     

Aqueous-phase quantitative NMR determination of amino acid enantiomer ratio by 13C-NMR using chiral neodymium shift reagent

A neodymium-(S)-PDTA (PDTA = N,N,N′,N′-tetrakis[(hydroxycarbonyl)methyl]-1,2-diaminopropane) complex was found exceptionally useful in the quantitative determination of enantiomer ratios of water-soluble natural amino acids by ¹³C-NMR. The method is demonstrated on mixtures of l- and d-enantiomers of various amino acids. The interactions of the chiral shift reagent with the amino acid molecules were rationalized by molecular orbital calculations.     

One-pot Microbial Synthesis of 2′-deoxyribonucleoside from Glucose, Acetaldehyde, and a Nucleobase

A one-pot enzymatic synthesis of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker’s yeast (Saccharomyces cerevisiae) generated ATP which was used to produce d-glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The d-glyceraldehyde 3-phosphate produced was transformed to 2′-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2′-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2′-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.     

Differential Transmembrane Diffusion of Triiodothyronine and Thyroxine in Liposomes: Regulation by Lipid Composition

How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3′triiodo l-thyronine and 3,5,3′,5′ tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2,4,6-trinitrobenzene sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (¹²⁵I) 3,5,3′triiodo l-thyronine and (¹²⁵I) 3,5,3′,5′ tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single exponential. Results obtained show that 3,5,3′ triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the contrary, 3,5,3′, 5′ tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer. Received: 1 June 1998/Revised: 14 October 1998     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

The impact of different chelating leaving groups on the substitution kinetics of mononuclear PtII(1,2- trans - R , R -diaminocyclohexane)(X–Y) complexes

A set of three oxaliplatin derivatives containing 1,2-trans-R,R-diaminocyclohexane (dach) as a spectator ligand and different chelating leaving groups X–Y, viz., [Pt(dach)(O,O-cyclobutane-1,1-dicarboxylate)], or Pt(dach)(CBDCA), [Pt(dach)(N,O-glycine)]⁺, or Pt(dach)(gly), and [Pt(dach)(N,S-methionine)]⁺, or Pt(dach)(l-Met), where l-Met is l-methionine, were synthesized and the crystal structure of Pt(dach)(gly) was determined by X-ray diffraction. The effect of the leaving group on the reactivity of the resulting Pt(II) complexes was studied for the nucleophiles thiourea, glutathione (GSH) and l-Met under pseudo-first-order conditions as a function of nucleophile concentration and temperature, using UV–vis spectrophotometric techniques. ¹H NMR spectroscopy was used to follow the substitution of the leaving group by guanosine 5′-monophosphate (5′-GMP²⁻) under second-order conditions. The rate constants indicate for all reactions a direct substitution of the X–Y chelate by the selected nucleophiles, thereby showing that the nature of the chelate, viz., O–O (CBDCA²⁻), N–O (glycine) or S–N (l-Met), respectively, plays an important role in the kinetic and mechanistic behavior of the Pt(II) complex. The k ₁ values for the reaction with thiourea, l-Met, GSH and 5′-GMP²⁻ were found to be as follows (10³ k ₁, 37.5 °C, M⁻¹ s⁻¹): Pt(dach)(CBDCA) 61 ± 2, 21.6 ± 0.1, 23 ± 1, 0.352 ± 0.002; Pt(dach)(gly) 82 ± 3, 6.2 ± 0.2, 37 ± 1, 1.77 ± 0.01; Pt(dach)(l-Met) (thiourea, GSH) 62 ± 2, 24 ± 1. The activation parameters for all reactions studied suggest an associative substitution mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gene cloning and overproduction of low-specificity d-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug

The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease. Received: 9 September 1999 / Received revision: 1 November 1999 / Accepted: 12 November 1999     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998     

Substrate specificity of a glucose-6-phosphate isomerase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> for monosaccharides

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.     

Enantiodifferent Proton Exchange in Alanine and Asparagine in the Presence of H 2 17 O

Using Time Domain ¹H Nuclear Magnetic Resonance with H₂¹⁷O (H₂¹⁷O-TD-¹HNMR), we found [H₂¹⁷O]- and pH-controlled chiral differences in proton exchange properties in alanine (Ala) and asparagine (Asn). To minimize and equalize chemical impurities, Asn enantiomers were purified by crystallization from racemic solution. At <0.1 M H₂¹⁷O, a shift in isoelectric pH (pI) occurred, ~1.14 kJ mol⁻¹ l-d-Asn ΔΔG ^o′ in the 5.91–6.42 pH range. One potential source for this asymmetry is the enantio-different magnetic moments (lμ↑ ≠ dμ↓) produced by neutral ring currents in the chiral center, leading to enantio-different nuclear spin organization and charge distribution in the amino group. At ≥pI, dissimilar interactions may occur in the hydration of the amino group with H₂¹⁷O (NH₂/H₂¹⁷O ≠ NH₂/H₂¹⁶O; NH₃ ⁺/H₂¹⁷O ≠ NH₂/H₂¹⁷O; l-*C-NH₂/H₂¹⁷O ≠ d-*C-NH₂/H₂¹⁷O). As lμ↑ ≠ dμ↓, the l-*C-amino and the d-*C-amino groups are diastereo spin-isomers. The nuclear spin of ¹⁷O may be parallel or antiparallel with the ortho-¹H¹H pair; hence two ortho-H₂¹⁷O molecules exist, also diastereo spin-isomers. As the pK of H₂¹⁷O is different from H₂¹⁶O, dissimilarities between l-*C- and d-*C-amino groups are converted into proton exchange differences. During H₂¹⁷O-TD-¹HNMR, the H₂¹⁷O molecule is a “probe” of the state of the amino group. Regarding prebiotic evolution: prebiotic chirality may not require stochastic symmetry breaking or preexisting chiral conditions; chemical chiral effects due to lμ↑ ≠ dμ↓ are small and need chiral amplification to generate an enantiomeric excess significant for prebiotic evolution; and prebiotic symmetry breaking was homochiral because the effect of lμ↑ and dμ↓ on the amino group should be similar in all alpha amino acids.