Mechanism of action of adenosylcobalamin: hydrogen transfer in the inactivation of diol dehydratase by glycerol

We have investigated the kinetic characteristics of the inactivation of the adenosylcobalamin-dependent enzyme propanediol dehydratase by glycerol, (RS)-1,1-dideuterioglycerol, (R)-1,1-dideuterioglycerol, and perdeuterioglycerol in the presence of 1,2-propanediol and 1,1-dideuterio-1,2-propanediol. The results imply that hydrogen (or deuterium) attached to C-1 of 1,2-propanediol participates in the inactivation process and contributes to the expression of a kinetic isotope effect on the rate of inactivation. The mechanism for this inactivation must involve the cofactor as an intermediate hydrogen carrier, presumably in the form of 5'-deoxyadenosine. Moreover, a mechanism involving a rate-determining transfer of hydrogen from an intermediate containing three equivalent hydrogens quantitatively accounts for all of the results. When diol dehydratase holoenzyme is inactivated by [1-3H]glycerol, 5'-deoxyadenosine which is enriched in tritium by a factor of 2.1 over that in glycerol can be isolated from the reaction mixture.     

Microbial metabolism of 1,2-propanediol studied by the Rumen Simulation Technique (Rusitec)

C zerkawski J.W., P iatkova M. & B reckenridge , G. 1984. Microbial metabolism of 1,2-propanediol studied by the Rumen. Simulation Technique (Rusitec). Journal of Applied Bacteriology , 56 , 81–94.
A series of experiments with the Rumen Simulation Technique (Rusitec) showed that 1,2-propanediol was metabolized efficiently by rumen micro-organisms and that the main end-products of fermentation were propionic and 2-methylbutyric acids. 'Propionaldehyde and n-propanol were also formed as intermediate compounds.' The effect of the diol on digestion of the basal diet appeared to be small with concentrate, or when the roughage was supplemented with additional nitrogen (urea). The decrease in the output of acetic and butyric acids was consistent with utilization of C₂ units for synthesis of 2-methylbutyric acid. The fermentation of 1,2-propanediol resulted in little or no increase in the output of additional microbial matter. The distribution of radioactivity from [1-¹⁴C] 1,2-propanediol confirmed that propionaldehyde and n -propanol were the primary products of metabolism of the diol and that the end-products were propionic and 2-methylbutyric acids, with very little labelling of microbial matter. Between 2% and 3% of radioactivity was found in gases and surprisingly the specific radioactivity of methane was higher than that of carbon dioxide, particularly during the initial stages of incubation. Possible pathways in the degradation of 1,2-propanediol by rumen micro-organisms are suggested and discussed in relation to similar reactions established in other systems.     

Recognition of damaged DNA by Escherichia coli Fpg protein: insights from structural and kinetic data

Formamidopyrimidine-DNA glycosylase (Fpg) excises oxidized purines from damaged DNA. The recent determination of the three-dimensional structure of the covalent complex of DNA with Escherichia coli Fpg, obtained by reducing the Schiff base intermediate formed during the reaction [Gilboa et al., J. Biol. Chem. 277 (2002) 19811] has revealed a number of potential specific and non-specific interactions between Fpg and DNA. We analyze the structural data for Fpg in the light of the kinetic and thermodynamic data obtained by the method of stepwise increase in ligand complexity to estimate relative contributions of individual nucleotide units of lesion-containing DNA to its total affinity for this enzyme [Ishchenko et al., Biochemistry 41 (2002) 7540]. Stopped-flow kinetic analysis that has allowed the dissection of Fpg catalysis in time [Fedorova et al., Biochemistry 41 (2002) 1520] is also correlated with the structural data.     

Inositol 1,2-cyclic 4,5-trisphosphate is formed in the rat parotid gland on muscarinic stimulation

Hawkins et al. [Hawkins, P.T., Berrie, C.P., Morris, A.J., and Downes, C.P. (1987) Biochem J. 243, 211-218] were unable to find any formation of inositol 1,2-cyclic 4,5-trisphosphate on muscarinic stimulation of rat parotid slices, contrary to what has been found in mouse pancreas and in platelets. We have repeated the studies of Hawkins et al. using [3H]inositol-prelabelled rat parotid minilobules and our improved HPLC method for clearly separating the three inositol trisphosphates. Substantial amounts of inositol 1,2-cyclic 4,5-trisphosphate formed on muscarinic stimulation of rat parotid minilobules, amounting to 5% of inositol 1,4,5-trisphosphate at 10 sec and one third of inositol 1,4,5-trisphosphate at 5 min.     

Identification of the 1,2-propanediol-1-yl radical as an intermediate in adenosylcobalamin-dependent diol dehydratase reaction

The reaction catalyzed by adenosylcobalamin-dependent diol dehydratase proceeds by a radical mechanism. A radical pair consisting of the Co(II) of cob(II)alamin and an organic radical intermediate formed during catalysis gives EPR spectra. The high-field doublet and the low-field broad signals arise from the weak interaction of an organic radical with the low-spin Co(II) of cob(II)alamin. To characterize the organic radical intermediate in the diol dehydratase reaction, several deuterated and (13)C-labeled 1,2-propanediols were synthesized, and the EPR spectra observed in the catalysis were measured using them as substrate. The EPR spectra with the substrates deuterated on C1 showed significant line width narrowing of the doublet signal. A distinct change in the hyperfine coupling was seen with [1-(13)C]-1,2-propanediol, but not with the [2-(13)C]-counterpart. Thus, the organic radical intermediate observed by EPR spectroscopy was identified as the 1,2-propanediol-1-yl radical, a C1-centered substrate-derived radical.     

Purification of an alanine racemase from Streptococcus faecalis and analysis of its inactivation by (1-aminoethyl)phosphonic acid enantiomers

An alanine racemase has been purified some 30 000-fold almost to homogeneity from Gram-positive Streptococcus faecalis NCIB 6459; the enzyme has been purified to the same extent (4000-fold) from an O-carbamyl-D-serine-resistant mutant with a 7-fold higher enzyme level in crude extract. The racemase has one pyridoxal phosphate molecule per 42-kDa subunit, has a Vmax of 3570 units/mg and a Km of 7.8 mM in the L to D direction, and has a Vmax of 1210 units/mg and a Km of 2.2 mM in the D to L direction. The Keq is 0.8 and kcat/Km values are ca. 3 X 10(5) M-1 s-1. The purified enzyme is inhibited in a time-dependent manner by both L- and D-(l-aminoethyl)phosphonates (Ala-P), confirming observations of Atherton et al. in crude extracts of this organism [Atherton, F. R., Hall, M. J., Hassal, C. H., Holmes, S. W., Lambert, R. W., Lloyd, W. J., & Ringrose, P. S. (1980) Antimicrob. Agents Chemother. 18, 897]. Studies with [1-2H]-, [1-3H]-, and [1,2-14C]Ala-P rule out enzymic activation and processing as the basis for irreversible inhibition. Thus, enzyme after exposure to [14C]Ala-P or [alpha-3H]Ala-P and gel filtration contains stoichiometric amounts of radioactive label, but denaturation quantitatively releases intact Ala-P into solution as revealed by high-performance liquid chromatography and cocrystallization with authentic material. The Ala-P isomers are slow binding inhibitors of this racemase as is the alpha,alpha'-dimethyl analogue but not the D or L isomers of the corresponding phosphinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reply to response to Dyck et al. (2007) on polar bears and climate change in western Hudson Bay by Stirling et al. (2008)

We address the three main issues raised by Stirling et al. [Stirling, I., Derocher, A.E., Gough, W.A., Rode, K., in press. Response to Dyck et al. (2007) on polar bears and climate change in western Hudson Bay. Ecol. Complexity]: (1) evidence of the role of climate warming in affecting the western Hudson Bay polar bear population, (2) responses to suggested importance of human–polar bear interactions, and (3) limitations on polar bear adaptation to projected climate change. We assert that our original paper did not provide any “alternative explanations [that] are largely unsupported by the data” or misrepresent the original claims by Stirling et al. [Stirling, I., Lunn, N.J., Iacozza, I., 1999. Long-term trends in the population ecology of polar bears in western Hudson Bay in relation to climate change. Arctic 52, 294–306], Derocher et al. [Derocher, A.E., Lunn, N.J., Stirling, I., 2004. Polar bears in a warming climate. Integr. Comp. Biol. 44, 163–176], and other peer-approved papers authored by Stirling and colleagues. In sharp contrast, we show that the conclusion of Stirling et al. [Stirling, I., Derocher, A.E., Gough, W.A., Rode, K., in press. Response to Dyck et al. (2007) on polar bears and climate change in western Hudson Bay. Ecol. Complexity] – suggesting warming temperatures (and other related climatic changes) are the predominant determinant of polar bear population status, not only in western Hudson (WH) Bay but also for populations elsewhere in the Arctic – is unsupportable by the current scientific evidence.The commentary by Stirling et al. [Stirling, I., Derocher, A.E., Gough, W.A., Rode, K., in press. Response to Dyck et al. (2007) on polar bears and climate change in western Hudson Bay. Ecol. Complexity] is an example of uni-dimensional, or reductionist thinking, which is not useful when assessing effects of climate change on complex ecosystems. Polar bears of WH are exposed to a multitude of environmental perturbations including human interference and factors (e.g., unknown seal population size, possible competition with polar bears from other populations) such that isolation of any single variable as the certain root cause (i.e., climate change in the form of warming spring air temperatures), without recognizing confounding interactions, is imprudent, unjustified and of questionable scientific utility. Dyck et al. [Dyck, M.G., Soon, W., Baydack, R.K., Legates, D.R., Baliunas, S., Ball, T.F., Hancock, L.O., 2007. Polar bears of western Hudson Bay and climate change: Are warming spring air temperatures the “ultimate” survival control factor? Ecol. Complexity, 4, 73–84. doi:10.1016/j.ecocom.2007.03.002] agree that some polar bear populations may be negatively impacted by future environmental changes; but an oversimplification of the complex ecosystem interactions (of which humans are a part) may not be beneficial in studying external effects on polar bears. Science evolves through questioning and proposing hypotheses that can be critically tested, in the absence of which, as Krebs and Borteaux [Krebs, C.J., Berteaux, D., 2006. Problems and pitfalls in relating climate variability to population dynamics. Clim. Res. 32, 143–149] observe, “we will be little more than storytellers.”     

Comparison of the data, analysis, and results of X-ray absorption studies of cytochrome c oxidase

Differences in the methods of analysis of X-ray absorption data used by Powers et al. [Powers, L., Blumberg, W. E., Chance, B., Barlow, C., Leigh, J., Jr., Smith, J., Yonetani, T., Vik, S., & Peisach, J. (1979) Biochim. Biophys. Acta 547, 520-538; Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] and Scott et al. [Scott, R., Schwartz, J., & Cramer S. (1986) Biochemistry 25, 5546-5555] are clarified. In addition, we compare the X-ray absorption data and results for resting cytochrome c oxidase reported by both groups using the same analysis method and conclude apart from any assumptions that the data are not identical.     

Growth on D-arabitol of a mutant strain of Escherichia coli K12 using a novel dehydrogenase and enzymes related to L-1,2-propanediol and D-xylose metabolism.

Escherichia coli K12 cannot grow on D-arabitol, L-arabitol, ribitol or xylitol (Reiner, 1975). Using a mutant of E. coli K12 (strain 3; Sridhara et al., 1969) that can grow on L-1,2-propanediol, a second-stage mutant was isolated which can utilize D-arabitol as sole source of carbon and energy for growth. D-Arabitol is probably transported into the bacteria by the same system as that used for the transport of L-1,2-propanediol. The second-stage mutant constitutively synthesizes a new dehydrogenase, which is not present in the parent strain 3. This enzyme, whose native substrate may be D-galactose, apparently dehydrogenates D-arabitol to D-xylulose, and its structural gene is located at 68.5 +/- 1 min on the E. coli genetic map. D-Xylulose is subsequently catabolized by the enzymes of the D-xylose metabolic pathway.     

Cloning and nucleotide sequence of the structural gene encoding for human tryptophanyl-tRNA synthetase.

A structural gene encoding bovine (b) tryptophanyl-tRNA synthetase (WRS) has recently been cloned and sequenced [Garret et al., Biochemistry 30 (1991) 7809-7817]. Using part of this sequence as a hybridisation probe we have cloned and sequenced a structural gene encoding human polypeptide highly homologous with two mammalian proteins, bWRS [Garret et al., Biochemistry 30 (1991) 7809-7817; EMBL accession No. X52113] and rabbit peptide chain release factor [Lee et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3508-3512]. Identification of the sequence encoding a human WRS is based on (i) the presence of 'HIGH' and 'KMSKS' structural motifs typical for class-I aminoacyl-tRNA synthetases [Eriani et al., Nature 347 (1990) 203-206]; (ii) coincidence of the number of SH groups per subunit estimated experimentally [Muench et al., Science 187 (1975) 1089-1091] and deduced from the cDNA sequence (six in both cases); (iii) close resemblance of two WRS polypeptides sequenced earlier [Muench et al., Science 187 (1975) 1089-1091] and the predicted structure in two different regions.     

Haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26: X-ray crystallographic studies of dehalogenation of brominated substrates

The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.     

Production and interconversion of 1,2-propanediol and hydroxyacetone by Escherichia coli

The anaerobic metabolism of marginally lethal levels of [13C]formaldehyde by Escherichia coli (K12, MU352, CRB, and CR63) was followed in vivo by 13C NMR. The products include 1,2-propanediol. Under aeration, the 1,2-propanediol is converted to hydroxyacetone. The hydroxyacetone is reconverted to 1,2-propanediol when aeration is stopped. The process can be cycled by varying the rate of aeration.     

Effects of substitution of tryptophan 412 in the substrate activation pathway of yeast pyruvate decarboxylase.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, located on the putative substrate activation pathway and linking E91 on the alpha domain with W412 on the gamma domain of the enzyme. While C221 on the beta domain is the residue at which substrate activation is triggered [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635; Baburina, I., et al. (1996) Biochemistry 35, 10249-10255], that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 [Baburina, I., et al. (1998) Biochemistry 37, 1235-1244; Baburina, I., et al. (1998) Biochemistry 37, 1245-1255], thence to E91 on the alpha domain [Li, H., and Jordan, F. (1999) Biochemistry 38, 9992-10003], and then on to W412 on the gamma domain and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600]. Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities about 4- and 10-fold lower than that of the wild-type enzyme. Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and acceptor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluorescence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state kinetic measurements. While the substrate activation is still prominent in the W412F variant, its level is very much reduced in the W412A variant, signaling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation. The fluorescence studies demonstrate that W412 is a relatively minor contributor to the well-documented fluorescence of apopyruvate decarboxylase in its native state. The information about the W412 variants provides strong additional support for the putative substrate activation pathway from C221 --> H92 --> E91 --> W412 --> G413 --> thiamin diphosphate. The accumulating evidence for the central role of the beta domain in stabilizing the overall structure is summarized.     

Redesign of coenzyme B(12) dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols

Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract ? Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).     

Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes.

The crystal structures of rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).     

Potentiometric monitoring of the redox state of brain structures of freely-moving rats in sleep-wake cycles

Variations of brain tissue redox state potential (E) of freely-moving white rats (300-350 g) in cycles of wakefulness (W), slow-wave sleep (SWS), and paradoxical sleep (PS) were measured by platinum electrodes symmetrically implanted into the frontal and occipital cortices and hippocampus. In addition, EMG of neck muscles and general motor activity of animals were recorded. The common reference electrode was implanted in the nasal bone. It was shown that in some brain sites (called active), episodes of W and PS were accompanied by a rise of E, and during transitions from W and PS to SWS, E dropped. The value of E varied in the range of 100 mV. It is suggested that transitions from W and PS to SWS are accompanied by shifts in the balance between the main energy sources. Oxidative phosphorylation prevails in W and PS, whereas aerobic glycolysis is the main source of energy during SWS. We think that this suggestion is supported both by a decrease in E in SWS and its oscillations typical of glucolytic processes [Aon et al., 1992]. Recent literature data [Bitter et al., 1996] suggest that astroglia is the main compartment for aerobic glycolysis.     

Bh-DNA: variations of the poly[d(A)].poly[d(T)] structure within the framework of the fibre diffraction studies

A refinement of the recent results for poly[d(A)].poly[d(T)] (Alexeev et al., J. Biomol. Struct. Dyn. 4,989 (1987)) involving additional parameters of the base-pair structure and of the sugar-phosphate backbone expands the conformational potential of this polynucleotide of the B type to include the possibility of bifurcated hydrogen bonds of the kind recently discovered in crystalline deoxyoligonucleotide with lone d(A)n.d(T)n stretch (Nelson et al., Nature 330, 221 (1987)). Still, analysis of the available data and energy calculations do not seem to indicate that the bifurcated H-bonds are a crucial factor responsible for the anomalous structure of the d(A)n.d(T)n sequence. The unique structural properties of poly[d(A)].poly[d(T)] can hardly be explained without taking into account its interactions with the double-layer hydration spine in the minor groove. In view of the hydration mechanism stabilizing poly[d(A)].poly[d(T)] and of the polynucleotide's heteronomous prehistory (Arnott et al., Nucleic Acids Res. 11,4141 (1983)) we suggest that this B-type structure be called Bh.     

Subunit positioning and transmembrane helix organisation in the core dimer of photosystem II

Recently 3D structural models of the photosystem II (PSII) core dimer complexes of higher plants (spinach) and cyanobacteria (Synechococcus elongatus) have been derived by electron [Rhee et al. (1998) Nature 396, 283-286; Hankamer et al. (2001) J. Struct. Biol., in press] and X-ray [Zouni et al. (2001) Nature 409, 739-743] crystallography respectively. The intermediate resolutions of these structures do not allow direct identification of side chains and therefore many of the individual subunits within the structure are unassigned. Here we review the structure of the higher plant PSII core dimer and provide evidence for the tentative assignment of the low molecular weight subunits. In so doing we highlight the similarities and differences between the higher plant and cyanobacterial structures.     

Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase

The three-dimensional structure of Wolinella succinogenes quinol:fumarate reductase (QFR), a dihaem-containing member of the superfamily of succinate:quinone oxidoreductases (SQOR), has been determined at 2.2 A resolution by X-ray crystallography [Lancaster et al., Nature 402 (1999) 377-385]. The structure and mechanism of W. succinogenes QFR and their relevance to the SQOR superfamily have recently been reviewed [Lancaster, Adv. Protein Chem. 63 (2003) 131-149]. Here, a comparison is presented of W. succinogenes QFR to the recently determined structure of the mono-haem containing succinate:quinone reductase from Escherichia coli [Yankovskaya et al., Science 299 (2003) 700-704]. In spite of differences in polypeptide and haem composition, the overall topology of the membrane anchors and their relative orientation to the conserved hydrophilic subunits is strikingly similar. A major difference is the lack of any evidence for a 'proximal' quinone site, close to the hydrophilic subunits, in W. succinogenes QFR.