Stereochemistry of meso-alpha,epsilon-diaminopimelate decarboxylase reaction: the first evidence for pyridoxal 5'-phosphate dependent decarboxylation with inversion of configuration

The stereochemistry of the decarboxylation of meso-alpha,epsilon-diaminopimelate catalyzed by meso-alpha,epsilon-diaminopimelate decarboxylase (EC 4.1.1.20) of Bacillus sphaericus was determined by stereochemical analyses of [6-2H]-L-lysine produced by the reaction in D2O. The product [6-2H]-L-lysine was converted to levorotatory methyl 5-phthalimido[5-2H]valerate by the reactions not affecting the absolute configuration of the asymmetric carbon atom. By contrast, methyl 5-phthalimido[5-2H]valerate derived from [2,6-2H2]-L-lysine, which was produced from [2,6-2H2]diaminopimelate by decarboxylation in H2O, was dextrorotatory. The authentic methyl (R)-5-phthalimido[5-2H]valerate prepared from L-glutamate with glutamate decarboxylase was levorotatory. These results indicate that the meso-alpha,epsilon-diaminopimelate decarboxylase reaction proceeds in an inversion mode. The deuterium label in [6-2H]-L-lysine was fully conserved during the conversion into pelletierine through [1-2H]cadaverine by the stereospecific diamine oxidase reaction. Thus, the enzymatic decarboxylation of meso-alpha,epsilon-diaminopimelate occurs with inversion of configuration in contrast to the other amino acid decarboxylase reported so far.     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ

Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris). Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme. Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases. The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM). The corresponding DD-isomers (3d and 6d) are less effective. The N-modified analogs are the most potent competitive inhibitors. The inhibition constant (Ki) values for B. sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively. These N-modified analogs do not effectively inhibit L-lysine decarboxylase. None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives. Possible mechanisms of inhibition are discussed. In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media. Other analogs (1----3) showed essentially no antibacterial activity.     

Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast

The steric courses of the reactions catalyzed by phosphatidylserine (PS) synthase from Escherichia coli and yeast were elucidated by the following procedure. RP and SP isomers of 1,2-dipalmitoyl-sn-glycero-3-[17O,18O]phosphoethanolamine ([17O,18O]DPPE) were synthesized with slight modification of the previous procedure [Bruzik, K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754] and converted to (RP)- and (SP)-1,2-dipalmitoyl-sn-glycero-3-[16O,17O,18O]phosphoric acid ([16O,17O18O]DPPA), respectively, by incubating with phospholipase D. Condensation of [16O,17O,18O]DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, [17O,18O]cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ([17O,18O]CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of [17O,18O]CDP-DPG with a mixture of L-serine, PS synthase (which converted [17O,18O]CDP-DPG to phosphatidylserine), and PS decarboxylase (which catalyzes decarboxylation of phosphatidylserine) gave [17O,18O]DPPE. The configuration and isotopic enrichments of the starting [17O,18O]DPPE and the product were analyzed by 31P NMR following trimethylsilylation of the DPPE. The results indicate that the reaction of E. coli PS synthase proceeds with retention of configuration at phosphorus, which suggests a two-step mechanism involving a phosphatidyl-enzyme intermediate, while the yeast PS synthase catalyzes the reaction with inversion of configuration, which suggests a single-displacement mechanism. Such results lend strong support to the ping-pong mechanism proposed for the E. coli enzyme and the sequential Bi-Bi mechanism proposed for the yeast enzyme, both based on previous isotopic exchange experiments.     

Stereochemistry of reactions catalyzed by glutamate decarboxylase.

When the decarboxylation of L-glutamic acid by the glutamate decarboxylase from Escherichia coli is carried out in D2O, the product gamma-aminobutyric acid contains a single deuterium atom. The stereochemistry of this material was established by conversion to levorotatory methyl 4-phthalimido [4(-2)H] butyrate. The dextrorotatory isomer of the latter compound was synthesized from S-[2(-2)H] glycine by a series of reactions not affecting the stereochemistry at the chiral center. Thus, the decarboxylation of glutamic acid occurs with retention of configuration. Decarboxylation of L-alpha-methylglutamic acid by this enzyme produced levorotatory gamma-aminovaleric acid and thus also occurs with retention of configuration.     

Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction

(Rp)- and (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphates have been synthesized by reaction of the respective (Sp)- and (Rp)-phosphorothioate precursors with N-bromosuccinimide in dioxane and H218O. Stereochemical analysis of the product derived from the (Rp)-phosphorothioate by digestion with snake venom phosphodiesterase in H217O and examination of the isotopic chirality of the resulting thymidine 5'-[16O,17O,18O]phosphate demonstrate that the replacement reaction has proceeded with inversion of configuration at phosphorus. Inspection of the 31P NMR spectrum of the methyl esters prepared from (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphate confirms that the replacement reaction has proceeded with very little if any racemization. This spectrum also allows the assignment of the absolute configuration of these methyl triesters. (Rp)-5'-O-Thymidyl 3'-O-thymidyl [18O]phosphate has been used to demonstrate that the stereochemical course of the hydrolytic reaction catalyzed by nuclease P1 from Penicillium citrum proceeds with inversion of configuration at phosphorus and therefore probably does not involve the participation of a covalent enzyme intermediate.     

Stereochemistry of the methylmalonyl-CoA decarboxylation reaction

The steric course of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA, catalyzed by the biotin-dependent sodium pump methylmalonyl-CoA decarboxylase of Veillonella alcalescens was determined. The decarboxylation of (S)-methylmalonyl-CoA in 3H2O yielded (R)-[2-3H]propionyl-CoA; and the decarboxylation of (S)-[2-3H]methylmalonyl-CoA in H2O produced (S)-[2-3H]propionyl-CoA. The results demonstrate retention of configuration during the decarboxylation reaction. The substrate stereochemistry of methylmalonyl-CoA decarboxylase is thus the same as that of all other biotin-containing enzymes investigated.     

Stereochemistry of ornithine decarboxylase reaction

To determine the steric course of the reaction of bacterial ornithine decarboxylase [EC 4.1.1.17], we have carried out the decarboxylation of L-ornithine in 2H2O and that of DL-[2-2H]ornithine in H2O, and obtained putrescine bearing a single deuterium atom in the C-1 position. The stereochemistry of [1-2H]putrescine was established by conversion to 1-(2-pyrrolidinyl)-2-propanone with acetoacetate and the pro-S hydrogen-specific diamine oxidase from pea seedlings. Analysis of deuterium content by gas chromatography-mass spectrometry showed that the deuterium label was fully retained during the conversion of [1-2H]putrescine produced by the decarboxylation of L-ornithine in 2H2O to 1-(2-pyrrolidinyl)-2-propanone, in contrast with the considerable loss of label from [1-2H]putrescine which was produced by the decarboxylation of DL-[2-2H]ornithine in H2O. The extent of loss of the deuterium label was in good agreement with the estimated value based on the isotope effect in the diamine oxidase reaction. These results indicate that the introduced deuterium (or hydrogen) is in the pro-R position at C-1 of putrescine, and consequently the ornithine decarboxylase reaction proceeds with retention of configuration.     

Stereochemical course of DNA hydrolysis by nuclease S1

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.     

The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction

The restriction endonuclease EcoRI hydrolyzes the Rp diastereomer of d(pGGsAATTCC), an analogue of d(pGGAATTCC) containing a chiral phosphorothioate group at the cleavage site between the deoxyguanosine and the deoxyadenosine residues (Connolly, B.A., Potter, B.V.L., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984) Biochemistry 23, 3343-3453). Performing the reaction in H2(18)O leads to d(pGG) and the hexanucleotide d([18O, S]pAATTCC) which has an 18O-containing phosphorothioate group at the 5' terminus. Further hydrolysis of this hexamer with nuclease P1 yields deoxyadenosine 5'-O-[18O]phosphorothioate which can be stereospecifically phosphorylated with adenylate kinase and pyruvate kinase to give Sp-[18O] deoxyadenosine 5'-O-(1-thiotriphosphate). 31P NMR spectroscopy shows the oxygen-18 in this compound to be in a bridging position between the alpha- and beta-phosphorus atoms. Thus, the hydrolysis reaction catalyzed by EcoRI proceeds with inversion of configuration at phosphorus. This result is compatible with a direct enzyme-catalyzed nucleophilic attack of H2O at phosphorus without involvement of a covalent enzyme intermediate.     

Steric course of the hydration of D-gluco-octenitol catalyzed by alpha-glucosidases and by trehalase

Crystalline Aspergillus niger alpha-glucosidase and highly purified preparations of rice alpha-glucosidase II and Trichoderma reesei trehalase were found to catalyze the hydration of [2-(2)H]-D-gluco-octenitol, i.e., (Z)-3,7-anhydro-1,2-dideoxy-[2-2H]-D-gluco-oct-2-enitol, to yield 1,2-dideoxy-[2-2H]-D-gluco-octulose. In each case, the stereochemistry of the reaction was elucidated by examining the newly formed centers of asymmetry at C-2 and C-3 of the hydration product. The C-1 to C-3 fragment of each isolated [2-2H]-D-gluco-octulose product was recovered as [2-2H]propionic acid and identified by its positive optical rotatory dispersion as the S isomer, showing that each enzyme had protonated the octenitol (at C-2) from above its re face. 1H NMR spectra of enzyme/D-gluco-octenitol digests in D2O showed that the alpha-anomer of [2-2H]-D-gluco-octulose was exclusively produced by each alpha-glucosidase, whereas the beta-anomer was formed by action of the trehalase. The trans hydration catalyzed by the alpha-glucosidases was found to be very strongly inhibited by the substrate; the cis hydration reaction catalyzed by the trehalase showed no such inhibition. Special importance is attached to the finding that in hydrating octenitol each enzyme creates a product of the same anomeric form as in hydrolyzing an alpha-D-glucosidic substrate. This result adds substantially to the growing evidence that individual glycosylases create the configuration of their reaction products by a means that is independent of donor substrate configuration, that is, by a means other than "retaining" or "inverting" substrate configuration.     

Phospholipids chiral at phosphorus. Stereochemical mechanism for the formation of inositol 1-phosphate catalyzed by phosphatidylinositol-specific phospholipase C.

The phosphatidylinositol-specific phospholipase C (PI-PLC) from mammalian sources catalyzes the simultaneous formation of both inositol 1,2-cyclic phosphate (IcP) and inositol 1-phosphate (IP). It has not been established whether the two products are formed in sequential or parallel reactions, even though the latter has been favored in previous reports. This problem was investigated by using a stereochemical approach. Diastereomers of 1,2-dipalmitoyl-sn-glycero-3-(1D- [16O,17O]phosphoinositol) ([16O,17O]DPPI) and 1,2-dipalmitoyl-sn-glycero-3-(1D-thiophosphoinositol) (DPPsI) were synthesized, the latter with known configuration. Desulfurization of the DPPsI isomers of known configurations in H2(18)O gave [16O,18O]DPPI with known configurations, which allowed assignment of the configurations of [16O,17O]DPPI on the basis of 31P NMR analyses of silylated [16O,18O]DPPI and [16O,17O]DPPI (the inositol moiety was fully protected in this operation). (Rp)- and (Sp)-[16O,17O]DPPI were then converted into trans- and cis-[16O,17O]IcP, respectively, by PI-PLC from Bacillus cereus, which had been shown to proceed with inversion of configuration at phosphorus [Lin, G., Bennett, F. C., & Tsai, M.-D. (1990) Biochemistry 29, 2747-2757]. 31P NMR analysis was again used to differentiate the silylated products of the two isomers of IcP, which then permitted assignments of IcP with unknown configuration derived from transesterification of (Rp)- and (Sp)-[16O,17O]DPPI by bovine brain PI-PLC-beta 1. The results indicated inversion of configuration, in agreement with the steric course of the same reaction catalyzed by PI-PLCs from B. cereus and guinea pig uterus reported previously. For the steric course of the formation of inositol 1-phosphate catalyzed by PI-PLC, (Rp)- and (Sp)-[16O,17O]DPPI were hydrolyzed in H2(18)O to afford 1-[16O,17O,18O]IP, which was then converted to IcP chemically and analyzed by 31P NMR. The results indicated that both B. cereus PI-PLC and the PI-PLC-beta 1 from bovine brain catalyze conversion of DPPI to IP with overall retention of configuration at phosphorus. These results suggest that both bacterial and mammalian PI-PLCs catalyze the formation of IcP and IP by a sequential mechanism. However, the conversion of IcP to IP was detectable by 31P NMR only for the bacterial enzyme. Thus an alternative mechanism in which IcP and IP are formed by totally independent pathways, with formation of IP involving a covalent enzyme-phosphoinositol intermediate, cannot be ruled out for the mammalian enzyme. It was also found that both PI-PLCs displayed lack of stereo-specifically toward the 1,2-diacylglycerol moiety, which suggests that the hydrophobic part of phosphatidylinositol is not recognized by PI-PLC.     

Studies on structure-function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway.

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207-1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.     

Mevalonate-5-diphosphate decarboxylase: stereochemical course of ATP-dependent phosphorylation of mevalonate 5-diphosphate

Chicken liver mevalonate-5-diphosphate decarboxylase catalyzes the reaction of mevalonate 5-diphosphate (MVADP) with ATP to produce isopentenyl diphosphate, ADP, CO2, and inorganic phosphate. The overall reaction involves an anti elimination of the tertiary hydroxyl and carboxyl groups. To investigate the mechanism for transfer of the terminal phosphoryl group of ATP to the C-3 oxygen of MVADP, we have carried out the reaction using stereospecifically labeled (Sp)-adenosine 5'-O-(3-thio[3-17O2,18O]triphosphate) [( gamma-17O2,18O]ATP gamma S) in place of ATP. The configuration of the [17O,18O]thiophosphate produced was found to be Rp, corresponding to overall inversion of configuration at phosphorus in the thiophosphoryl group transfer step. This result is consistent with the direct transfer of the thiophosphoryl group from (Sp)-[gamma-17O2,18O]ATP gamma S to MVADP at the active site. Our result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.     

Stereospecificity of malonyl-CoA decarboxylase, acetyl-CoA carboxylase, and fatty acid synthetase from the uropygial gland of goose

Malonyl-CoA decarboxylase from the uropygial gland of goose decarboxylated (R,S)-methylmalonyl-CoA at a slow rate and introduced 3H from [3H]2O into the resulting propionyl-CoA. Carboxylation of this labeled propionyl-CoA by propionyl-CoA carboxylase from pig heart and acetyl-CoA carboxylase from the uropygial gland completely removed 3H. Repeated treatment of (R,S)-[methyl-14C]methylmalonyl-CoA with the decarboxylase converted 50% of the substrate into propionyl-CoA, whereas (S)-methylmalonyl-CoA, generated by both carboxylases, was completely decarboxylated. Radioactive (R)- (S), and (R,S)-methylmalonyl-CoA were equally incorporated into fatty acids by fatty acid synthetase from the uropygial gland. The residual methylmalonyl-CoA remaining after fatty acid synthetase reaction on (R,S)-methylmalonyl-CoA was also racemic. These results show that: (a) the decarboxylase is stereospecific, (b) replacement of the carboxyl group by hydrogen occurs with retention of configuration, (c) acetyl-CoA carboxylase of the uropygial gland generates (S)-methylmalonyl-CoA from propionyl-CoA, and (d) fatty acid synthetase is not stereospecific for methylmalonyl-CoA.     

Meso-alpha,epsilon-diaminopimelate D-dehydrogenase: distribution and the reaction product.

A high activity of meso-alpha-epsilon-diaminopimelate dehydrogenase was found in extracts of Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, and Proteus vulgaris among bacteria tested. B. sphaericus IFO 3525, in which the enzyme is most abundant, was chosen to study the enzyme reaction. The enzyme was not induced by the addition of meso-alpha-epsilon-diaminopimelate to the growth medium. The reaction product was isolated and identified as alpha-amino-epsilon-ketopimelate by a comparison of the properties of its 2,4-dinitrophenylhydrazone with those of an authentic sample in silica gel thin-layer chromatography, absorption, infrared and proton nuclear magnetic resonance spectrometry, and elemental analyses. The alpha-amino-epsilon-ketopimelate formed enzymatically was decarboxylated by H2O2 to yield L-alpha-aminoadipate. This suggests that the amino group with D-configuration in the substrate is oxidatively deaminated; the enzyme is a D-amino acid dehydrogenase. L-alpha-Amino-epsilon-ketopimelate undergoes spontaneous dehydration to the cyclic delta1-piperideine-2,6-dicarboxylate. The enzyme reaction is reversible, and meso-alpha-epsilon-diaminopimelate was formed in the reductive amination of L-alpha-epsilon-ketopimelate.     

Phosphoglycerate mutase from wheat germ: studies with isotopically labeled 3-phospho-D-glycerates showing that the catalyzed reaction is intramolecular. Appendix: phosphoglycerate mutase from wheat germ: isolation, crystallization, and properties.

The isomerization of 3-phospho-D-glycerate and 2-phospho-D-glycerate catalyzed by the cofactor-independent phosphoglycerate mutase from wheat germ (the isolation and crystallization of which is described in the Appendix) has been shown to be intramolecular by two methods. Mass-spectrometric analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[18O]phospho-D-glycerate shows that there is no exchange of labeled phosphoryl group between carbon skeletons in the mutase-catalyzed reaction. Analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[32p]phospho-D-glycerate by a method involving the kinetic discrimination between 2(-2)H and 2(-1)H species using the enolase isotope effect similarly shows that the wheat germ phosphoglycerate mutase mediates an intramolecular transfer of the phosphoryl group.     

The stereochemical course of the ribulose-5-phosphate kinase-catalyzed reaction

Spinach-leaf ribulose-5-phosphate kinase catalyzes the reaction of (Rp)-[beta, gamma-18O, gamma-18O]adenosine 5'-(3-thiotriphosphate) with ribulose 5-phosphate to form ribulose 1-[18O]phosphorothioate 5-phosphate. This product is incubated with CO2, Mg2+, and ribulose-bisphosphate carboxylase to form the [18O]phosphorothioate of D-glycerate. Reduction of this material using phosphoglycerate kinase/ATP, glyceraldehyde-3-phosphate dehydrogenase/NADH, triose-phosphate isomerase, and glycerol-phosphate dehydrogenase/NADH produces glycerol 3-[18O]phosphorothioate, which is subjected to ring closure using diethylphosphorochloridate. This in-line reaction produces a diastereoisomeric mixture of glycerol 2,3-cyclic phosphorothioates. 31P NMR spectroscopy was used to analyze the 18O content of the products. The anti-diastereoisomer, which is the major isomer formed and corresponds to the downfield 31P NMR signal (Pliura, D.H., Schomburg, D., Richard, J.P., Frey, P.A., and Knowles, J.R. (1980) Biochemistry 19, 325-329), retains the 18O label. This observation indicates that the ribulose-5-phosphate kinase reaction proceeds with inversion of configuration at phosphorus. The reaction is, therefore, unlikely to involve the participation of a covalent phosphoryl-enzyme intermediate.     

Stereochemistry of Decarboxylation Reactions Catalyzed by a Constitutive Aromatic l-Amino Acid Decarboxylase

The stereochemistry of the decarboxylation reaction catalyzed by an aromatic l-amino acid decarboxylase, purified from Micrococcus percitreus, was studied using stereospecifically deuterium labelled phenylalanine (Phe). The ¹H NMR spectrum of [1,2-²H₂]-β-phenethylamine enzymatically derived from (2S, 3R)-[3-²H]-Phe in ²H₂O was compared with that of [1-²H]-β-phenethylamine from unlabelled Phe in ²H₂O. The results clearly indicate that the decarboxylation reaction of this enzyme proceeds exclusively through a course in which the configuration at C-2 of Phe is retained.     

Gentamicin nucleotidyltransferase. Stereochemical inversion at phosphorus in enzymatic 2'-deoxyadenylyl transfer to tobramycin

Gentamicin nucleotidyltransferase-catalyzed reaction of (Sp)-[alpha-17O]dATP with tobramycin produced 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. The configuration at phosphorus in this product was shown to be Rp by chemical degradation to chiral [17O, 18O]dAMP using a stereochemically defined procedure, and determination of the configuration at phosphorus in this product. Periodate-base treatment of 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin followed by NaBH4 reduction produced (2-glyceryl)-[17O]dAMP, which upon snake venom phosphodiesterase-catalyzed hydrolysis in H(2)18O produced [17O,18O] dAMP. The configuration at phosphorus in this product was shown to be S by enzymatic phosphorylation to [17O,18O]dATP, adenylylcyclase (Bordetella pertussis)-catalyzed cyclization to 3',5'-cyclic [17O,18O]dAMP, and 31P NMR analysis of the ethyl esters. Since snake venom phosphodiesterase-catalyzed hydrolyses proceed with retention of configuration at phosphorus, (Sp)-[17O,18O]dAMP must have been produced from (Rp)-(2-glyceryl)-[17O]dAMP; and since the chemical degradation to the latter compound did not involve cleavage of any bonds to phosphorus, the initial enzymatic product must have been (Rp)-2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. Therefore, nucleotidyl transfer catalyzed by gentamicin nucleotidyl-transferase proceeds with inversion of configuration at phosphorus, and the reaction mechanism involves an uneven number of phosphotransfer steps. Inasmuch as this is an uncomplicated two-substrate group transfer reaction, the mechanism probably involves direct nucleotidyl transfer from the nucleoside triphosphate to the aminoglycoside. The B. pertussis adenylylcyclase reaction was shown to proceed with inversion at phosphorus, as has been established for other adenylylcyclases.