Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase

Abstract

Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B6, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5’-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24, and Arg215) that form an “arginine cage” and efficiently trap PLP. The crystal structure of the PNPO–PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate–PLP ternary complex catalytically incompetent. Site-directed mutagenesis studies focused on the arginine cage validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R21L variant). Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B6, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5’-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24, and Arg215) that form an “arginine cage” and efficiently trap PLP. The crystal structure of the PNPO–PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate–PLP ternary complex catalytically incompetent. Site-directed mutagenesis studies focused on the arginine cage validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R21L variant). Pyridoxal 5’-phosphate (PLP) acts as cofactor for over 150 enzymes (1Percudani R. Peracchi A. A genomic overview of pyridoxal-phosphate-dependent enzymes.EMBO Rep. 2003; 4: 850-854Crossref PubMed Scopus (326) Google Scholar, 2Percudani R. Peracchi A. The B6 database: A tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families.BMC Bioinformatics. 2009; 10: 273Crossref PubMed Scopus (166) Google Scholar) involved in a number of metabolic pathways such as the synthesis, transformation, and degradation of amines and amino acids; supply of one carbon units; transsulfuration; synthesis of tetrapyrrolic compounds (including heme) and polyamines; and biosynthesis and degradation of neurotransmitters. In the cell, PLP is supplied to apoenzymes through either biosynthesis or recycling of B6 vitamers coming from the environment and protein turnover. The latter route is the only one available in organisms that cannot synthesize PLP, such as humans, and they obtain it through a salvage pathway (Fig. 1) catalyzed by the enzymes pyridoxal kinase, pyridoxine 5’-phosphate oxidase (PNPO; EC 1.4.3.5), and either specific or nonspecific phosphatases (3di Salvo M.L. Contestabile R. Safo M.K. Vitamin B(6) salvage enzymes: Mechanism, structure and regulation.Biochim. Biophys. Acta. 2011; 1814: 1597-1608Crossref PubMed Scopus (137) Google Scholar). By using FMN as cofactor, PNPO catalyzes the oxidation of both pyridoxine 5’-phosphate (PNP) and pyridoxamine 5’-phosphate to PLP, reducing molecular oxygen to hydrogen peroxide. With the Escherichia coli PNPO (ePNPO), the specificity constant for PNP is 50-fold higher than that for pyridoxamine 5’-phosphate (4Zhao G. Winkler M.E. Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12.J. Bacteriol. 1995; 177: 883-891Crossref PubMed Google Scholar). In E. coli, which synthesizes PLP through the so-called DXP-dependent pathway, ePNPO (encoded by the pdxH gene) plays the dual role of salvage pathway enzyme and of last enzyme in PLP biosynthesis (Fig. 1). This is the reason why the reaction catalyzed by ePNPO is a key point in PLP homeostasis. We have recently demonstrated (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar) that ePNPO undergoes an allosteric feedback inhibition by PLP, through a mechanism in which binding of substrate at the active site and binding of PLP at an allosteric site to a (Fig. a strong allosteric coupling is also in the of of the ternary complex in and in the of by the complex in which PLP is bound at the allosteric to a PLP molecule at the active site in the of PLP concentration in binding of PLP at the allosteric site higher than binding at the active site, PLP binding at the active site of the enzyme PLP allosteric inhibition is also observed PNPO A. di Salvo M.L. Contestabile R. Tramonti A. of pyridoxine 5'-phosphate oxidase and its Rep. 10: PubMed Scopus Google Scholar). in the coupling between the allosteric site and the active site is that the complex a and PLP can also at the active site, an enzyme complex PLP in The PLP allosteric site of ePNPO to the binding by binding of pyridoxal 5'-phosphate to Escherichia coli pyridoxine 5'-phosphate Biophys. PubMed Scopus Google Scholar) they observed the enzyme is PLP and through a it PLP. This is to the that PLP at a site, from the active site, as demonstrated by a ePNPO active site is and that (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar). In the cell, PLP bound to site of ePNPO is from the environment to the of its it of amines and in the at the it can be to that ePNPO a role in the intracellular PLP binding of pyridoxal 5'-phosphate to Escherichia coli pyridoxine 5'-phosphate Biophys. PubMed Scopus Google Scholar). A crystal structure of ePNPO from in a a concentration of PLP a site located at the protein from the active site M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar). the of binding site to the PLP binding site In crystal in which PLP is also bound at the active site, PLP at the site is in an of and In the higher the PLP is between the of and the and the the amino of M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar) (Fig. PLP in the a the protein and from crystal M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar). Here we through mutagenesis that site is involved in the allosteric binding of PLP. the we a ePNPO an active site, to obtain a crystal structure of the enzyme in which PLP is bound at a site. We that the allosteric PLP binding site. The of the site by crystallographic studies M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar) in the allosteric binding of PLP through A and and of ePNPO (Fig. and to and to that the the structure of the (Fig. that in the structure caused by the to and of and The (Fig. to a 1) to obtain The enzyme a of The form a the of the is 1). that the the structure of the from the of ePNPO in from in from from PLP binding from PLP in from from PLP binding from PLP in a The properties of the ePNPO in at using PNP as In the PLP an complex at is to the of the PNPO reaction and properties of and PubMed Google Scholar). in PLP in the and the enzyme by binding at the allosteric site (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar). as a of substrate concentration using the and in the enzymes to that of in in the of the is than that of it is that the the properties of the of PLP to ePNPO also using the for the enzyme (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google which is on the of FMN we have demonstrated (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google PLP at the active site of binding at the allosteric site, which higher a that binding at the active site. the only binding of PLP at the allosteric site. PLP binding ePNPO (Fig. to the constant 1) that higher than that for ePNPO (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar). This is a role for the residues in PLP binding at the allosteric site. is by on ePNPO the protein is PLP and through an it PLP bound to it binding of pyridoxal 5'-phosphate to Escherichia coli pyridoxine 5'-phosphate Biophys. PubMed Scopus Google Scholar). This the active site is to PLP as a of (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google that PLP at an allosteric site. ePNPO an amount of PLP. they through an and the of PLP to protein subunits The of PLP and the and ePNPO 1). to that the enzyme that the site by crystallographic studies to the allosteric binding site. in the a crystal structure of ePNPO in which PLP is bound at both the active site and a site by M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar). This structure by the in a of PLP it be that PLP to the allosteric site, the site by Safo and M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar) and the active it is that the allosteric site in crystallographic unless the allosteric site by crystal We ePNPO and PLP in we either obtain or in which PLP bound at the active site we to and a form of ePNPO in which the active site is and to PLP. We recently that an ePNPO which a to PNP at the active site, is to PLP at the allosteric site (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar). We in both the of PLP in and of PLP in The of both crystal The of to the a the is by one FMN one and The of the by the crystallographic The of the to the a one FMN molecule one in one PLP molecule in and The the structural of the from to and In in the the is also in an residues The structure can be as by Safo M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase FMN at A PubMed Scopus Google Scholar) in of of the is by a and a by the that is of both The is by in on one and on the The to the and and is by the three and (Fig. In the the A a of residues residues corresponding to the ePNPO the and only a of three residues is in which be of the of from the (Fig. PLP is at the active site of either (Fig. A and in the of PLP for is a of the active site (Fig. and In to the the is the characterized structure of the enzyme in a in which the is This structural in the in a of and the and and The of in A to as a of PLP binding in a in at the interface between subunits A and mainly circumscribed by three arginine residues (Arg23, Arg24, Arg215) to and as an cage” for PLP. The three residues efficiently trap PLP in the (Fig. A and The binding is by the as in of the binding of pyridoxal PubMed Scopus Google which is of the amino of the and of and Arg24, In a of hydrogen between and the PLP by means of a role is to to PLP in the is by the to and the This cage by Safo and M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar) as a binding site for a the PLP and to in a very as only a of is in the binding site. The between the corresponding at (Fig. to the of a than PLP in of of the The of and between the the structural by PLP binding to the allosteric site. PLP binding the of a specific of hydrogen and of A to a protein of residues to of A and In structure of PLP, the between key residues of and and residues and on the to to structural and (Fig. of PLP to A of a of the structural of from its the of structural of which its structure and (Fig. The of PLP binding also the of on in the ePNPO structure to the PLP binding to the site, both its and in in the the a to that of the PLP binding to the site, and its its to its (Fig. The of the to and in the of the structure of the active site and the for both PLP and and as a the of the the amino residues Arg24, and for the PLP in the crystal and as as and and a ePNPO through mutagenesis and to that of the ePNPO (Fig. that the the structure of the from that to the enzyme to (Fig. 1). With enzyme and in to of ePNPO they to of the enzyme PLP binding through the (Fig. the PLP binding properties of to be as by that in to higher than that of the enzyme 1). ePNPO PLP and through an a of PLP only to to protein in the of the R23L/R215L and in the ePNPO 1). of PNP oxidation PLP catalyzed by PNPO in to PLP and enzyme inhibition and properties of and PubMed Google Scholar). by the reaction is in a complex of PLP formation is observed as a of PLP allosteric inhibition (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar). enzyme the of formation in a caused by the of PLP in the binding of PLP at the allosteric site, and the of enzyme inhibition (Fig. The is by a of PLP. This complex inhibition to the in which is that of an allosteric of PNP oxidation catalyzed by and in and and in in enzymes very to the enzyme, a of by a of the reaction only substrate the in PLP inhibition is in to In the R23L/R215L and PLP in is (Fig. and A of PLP inhibition the R23L/R215L and in The of PLP as a of PNP concentration at PLP to to the in to obtain and as a of PLP With ePNPO (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google PLP both and of and as a of PLP concentration a in which the enzyme is at PLP concentration the ternary complex, is is the R23L/R24L ePNPO a a (Fig. This that an PLP concentration to a in which the complex is catalytically a as the the of that PLP the enzyme by binding at at the active site and at an allosteric site, a A is observed the R23L/R215L In is by PLP that the complex a marked (Fig. the a inhibition to PLP, in which is constant and an on PLP concentration (Fig. that PLP the enzyme by binding only at the active site, the substrate In to the a inhibition (Fig. in which PLP at both the active and allosteric of PLP at the allosteric site and binding of substrate at the active site and by an binding of PLP at the active site and at the allosteric site the of the by a In PLP binding at the active site to complete is only PLP at the allosteric site is by a from to 1). In to be of the enzyme substrate and PLP, and of substrate to be from to in and enzymes PLP binding is and (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google the constant of the substrate binding be to to the constant from the of and on substrate and PLP concentration on the of as a of substrate at PLP the of and as a of PLP of the R23L/R24L by to from to which between and This in a in which very the (Fig. and and the for the and This is caused by a number of By and at the in the to by the that the in the the R23L/R24L and and higher than for in of in of for the in in a from of PLP inhibition ePNPO and to by in the to to by (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar). in a for the in in the to With the R23L/R215L the in a and and to and the for to and very The very that the complex is (Fig. and why the is to a of the either to or to and the in the a in which to and to a and and to the in the A (Fig. and which to the by the in the of the it is that PLP acts as a the of inhibition to by and to a and to In a of and (Fig. In to the PLP binding to ePNPO from the of inhibition the PLP binding using the the R23L/R24L and the of ePNPO and PLP a of of PLP protein R23L/R24L ePNPO and of one molecule of PLP protein ePNPO (Fig. mutagenesis studies that the site by crystallographic M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar) to the PLP binding site, and have for PLP binding and to PLP and the of the that the cage in crystallographic studies R23L/R215L and a of PLP by a of and a of PLP of the PLP binding site be to in a such as the complete of PLP the that PLP also to site, such as the active site, be and it a very as by inhibition and PLP binding studies that the cage to the PLP binding allosteric site. In the enzyme is by a strong allosteric coupling between binding PLP binding at the allosteric site and substrate binding at the active site. The of is in the of the of and in the of of the ternary complex in is a of structural on the protein by PLP and The of PLP binding at the allosteric site is in the crystal structure of PLP binding at the cage in the of a structural by a of and the and and it the which is a very structural of the active site. In the is from FMN and its to that of that it the of the and which is in PNPO from is involved in substrate binding and for a and M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar). The of the PNP to the FMN and its that the substrate is for an and of the hydrogen to In E. coli the in an in and a in Salvo M.L. Safo M.K. site structure and of Escherichia coli PubMed Scopus Google Scholar). The is also involved in substrate binding by the M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar). the structural observed in the allosteric of PLP on PNP binding at the active site of and also the of the By the it is that PNP binding at the active site, and the of the substrate and of the to PLP binding at the allosteric site. it be that the structure of the is in that PLP is bound only to one and the structural and and the to to that the it is why binding of PLP to both allosteric of the is in the of PLP binding to the allosteric site of ePNPO is of one PLP molecule protein (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google that the allosteric of PLP binding to one to the the binding of a PLP the the ePNPO an active is to PLP (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google that the of the active site caused by the have the allosteric of PLP binding at the allosteric site. the of the structure from the of the protein that that Kinetic inhibition studies provide the have the to the allosteric site and the active site to In to the R23L/R24L is of binding PLP at both the allosteric site and the active site (Fig. as also demonstrated by a PLP binding of PLP enzyme (Fig. This is the allosteric coupling between the is as also in the that the ternary complex of the PLP binding at the allosteric site the active site structure as as in the PLP binding at the active site is by PLP binding at the allosteric site is a as by a we observed a PNPO A. di Salvo M.L. Contestabile R. Tramonti A. of pyridoxine 5'-phosphate oxidase and its Rep. 10: PubMed Scopus Google Scholar) (Fig. in to A is observed the R23L/R215L In from binding of either PLP or substrate to the the enzyme can only PLP at the allosteric the complex is (Fig. cannot the substrate it is of binding a PLP molecule at the active site, the the the enzyme bound the substrate at the active site, it is of binding PLP at the allosteric site, it is to the of the substrate the of three of the cage residues PLP binding at the allosteric site, or at allosteric of PLP that PLP can only at the active site. This is from the inhibition observed the (Fig. a that is to the in the studies of PLP binding to the enzyme In the of for PLP binding and the of PLP observed the (in and as to the complete of PLP by the in PLP at the active site The allosteric of PLP is either or and as from a constant as a of PLP concentration (Fig. in the PLP at the allosteric site the complex cannot be of PLP formation in and (Fig. also the loss of the allosteric With both and in is as to that observed and R23L/R24L ePNPO that a to PLP allosteric for and for protein from which from 5'-phosphate from PLP to the of and Salvo M.L. Safo M.K. site structure and of Escherichia coli PubMed Scopus Google Scholar). of the pdxH using the as and a in the from by The and the E. coli to the by of both and the only to using the E. coli Salvo M.L. Safo M.K. site structure and of Escherichia coli PubMed Scopus Google Scholar). R23L/R24L R23L/R24L The E. coli Salvo M.L. Safo M.K. site structure and of Escherichia coli PubMed Scopus Google Scholar) for the protein The for the of the is a The in the between and The E. coli in and at to an of the of ePNPO and the for at and in The by of and on and the using the and the as Salvo E. G. Winkler M.E. and of Escherichia coli pyridoxine 5'-phosphate PubMed Scopus Google Scholar). The at through the the of the by and as Salvo E. G. Winkler M.E. and of Escherichia coli pyridoxine 5'-phosphate PubMed Scopus Google Scholar). The protein concentration of both and the of FMN to the at using the by di Salvo Salvo E. G. Winkler M.E. and of Escherichia coli pyridoxine 5'-phosphate PubMed Scopus Google Scholar). using a In a protein in 150 and in a of in a from to in The at to and the between and in using the of of the to of and to a 1) to obtain the the by the of the as a of by using the The for PLP binding to and enzymes of FMN observed binding of molecule to the PNPO allosteric binding site (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar). at in at a using a ePNPO at a concentration of of PLP to and at and from FMN as a of PLP concentration and using a in which is the is in the of is at is the PLP for the enzyme and is the constant of the to the of PLP binding using of enzyme and using the and at and using by at the from as to obtain an of using a using a at in at of a protein concentration of ePNPO for the R23L/R24L PNPO protein PNP concentration from to in the of a FMN concentration to that for ePNPO form by the enzyme at FMN formation at the a of and properties of and PubMed Google Scholar). The of and from of as a of PNP concentration to the of the and also in at In PLP formation as a of using an of at (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google using PLP concentration in of of vitamin Scopus Google enzyme and PNP and by the of the enzyme to PNP and constant by a to Kinetic the the of of reaction by PNP in PLP formation at using a of by PNP at a PLP concentration using the and which to and as a of PLP In to and and an on the of the PLP inhibition in for which the as a of the PNP substrate and the PLP The and as a of PLP and as a of PLP and to of PLP by ePNPO by the PLP of the protein PLP and The PNPO PLP and at for an PLP from the protein on a using a The the PLP bound to the protein which the PLP in the PNPO from the of E. coli as (5Barile A. Tramonti A. di Salvo M.L. Contestabile R. feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia PubMed Scopus Google Scholar, Salvo M.L. A. Tramonti A. R. Contestabile R. oxidase and the PubMed Scopus Google Scholar). the in 150 to a concentration of and for at at to protein at the by and by the protein to of both ePNPO and ePNPO in complex PLP. PLP to a protein in a the a of to and three of protein to to in three of in a to of a crystal for the and the the as a in a for to of a the as a in a to of of on and by in a and in and to the at from of and at using a at the of and and and available in and by molecular The crystal structure of of and as the in the the the crystallographic which the The and in the the A. A. Tramonti A. A. structure of the of from E. Scholar) and A. A. Tramonti A. A. structure of the of from E. coli in complex This M.K. di Salvo M.L. structure of Escherichia coli pyridoxine 5'-phosphate oxidase pyridoxal 5'-phosphate at A PubMed Scopus Google Scholar). The that they have of the of The to for and the of for crystal and A. and and A. R. and and and A. and and A. and and R. and and This by from to R. from di and to R. and the to A.

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