Substrate Specificity of Human Nucleoside-diphosphate Kinase Revealed by Transient Kinetic Analysis

Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of γ-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 × 106m−1s−1. Dephosphorylation by NDPs was 2–3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3–4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5′-O-(thiotriphosphate) (ATPγS) and guanosine 5′-O-(thiotriphosphate) (GTPγS) to NDP, but at 11000 of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATPγS and GTPγS occurred withkmax of 2.8 and 1.35 s−1 and Kd of 145 and 36 μm respectively. For dethiophosphorylation by a range of NDPs, kmaxwas in the range of 5–30 s−1, whereasKd varied between 0.16 and 3.3 mm. Guanine had the lowest Kd values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2′-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3′-ribose. Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of γ-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 × 106m−1s−1. Dephosphorylation by NDPs was 2–3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3–4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5′-O-(thiotriphosphate) (ATPγS) and guanosine 5′-O-(thiotriphosphate) (GTPγS) to NDP, but at 11000 of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATPγS and GTPγS occurred withkmax of 2.8 and 1.35 s−1 and Kd of 145 and 36 μm respectively. For dethiophosphorylation by a range of NDPs, kmaxwas in the range of 5–30 s−1, whereasKd varied between 0.16 and 3.3 mm. Guanine had the lowest Kd values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2′-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3′-ribose. Nucleoside-diphosphate kinase (NDK 1The abbreviations used are: NDK, nucleoside-diphosphate kinase; ATPγS, adenosine 5′-O-(thiotriphosphate); GTPγS, guanosine 5′-O-(thiotriphosphate); NTPγS, nucleoside 5′-O-(3-thiotriphosphate); GDPβS, guanosine 5′-O-2-(thio)diphosphate; ADPβS, adenosine 5′-O-2-(thio)diphosphate; NDPβS, nucleoside 5′-O-2-(thio)diphosphate; AZT-TP, 3′-Azido-3′-deoxythymidine triphosphate; AZT-DP, 3′-Azido-3′-deoxythymidine diphosphate; HPLC, high pressure liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; NDK-BHis∼thioP, thiophosphorylated NDK-BHis. 1The abbreviations used are: NDK, nucleoside-diphosphate kinase; ATPγS, adenosine 5′-O-(thiotriphosphate); GTPγS, guanosine 5′-O-(thiotriphosphate); NTPγS, nucleoside 5′-O-(3-thiotriphosphate); GDPβS, guanosine 5′-O-2-(thio)diphosphate; ADPβS, adenosine 5′-O-2-(thio)diphosphate; NDPβS, nucleoside 5′-O-2-(thio)diphosphate; AZT-TP, 3′-Azido-3′-deoxythymidine triphosphate; AZT-DP, 3′-Azido-3′-deoxythymidine diphosphate; HPLC, high pressure liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; NDK-BHis∼thioP, thiophosphorylated NDK-BHis.; EC 2.7.4.6) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate (1Weaver R.H. Enzymes. 1962; 6: 151-160Google Scholar), involving a phosphohistidine intermediate state (2Mourad N. Parks R.E. J. Biol. Chem. 1966; 241: 271-278Abstract Full Text PDF PubMed Google Scholar). NDK exists as either hexamers (most eukaryotic species) or tetramers (most prokaryotic species) and has been reported to display little specificity for different nucleotide bases (2Mourad N. Parks R.E. J. Biol. Chem. 1966; 241: 271-278Abstract Full Text PDF PubMed Google Scholar, 3Dumas C. Lascu I. Morera S. Glaser P. Fourme R. Wallet V. Lacombe M.-L. Veron M. Janin J. EMBO J. 1992; 11: 3203-3208Crossref PubMed Scopus (176) Google Scholar), reflecting a pivotal role in maintaining balanced levels of all oxy- and deoxynucleoside triphosphates in the cell. It is thus considered a housekeeping enzyme (4Parks R.E. Agarwal R.P. Enzymes. 1973; 8: 307-334Crossref Scopus (357) Google Scholar), a role that has taken on greater significance with the therapeutic use of nucleotide analogues such as azidothymidine (AZT) as replication inhibitors as these compounds must be phosphorylated in the cell before becoming active (5Furmann P.A. Fyfe J.A. St. Clair M.H. Weinhold K. Rideout J.L. Freeman G.A. Lehrman S.N. Bolognesi D.P. Broder S. Mitsuya H. Barry D.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8333-8337Crossref PubMed Scopus (1227) Google Scholar). In humans, two very closely (89% identity) related isoforms of NDK, designated NDK-A and NDK-B, were identified biochemically (6Gilles A.M. Presecan E. Vonica A. Lascu I. J. Biol. Chem. 1991; 266: 8784-8789Abstract Full Text PDF PubMed Google Scholar), which associate in vivo to form mixed hexameric isoenzymes. Evidence that NDK-A and -B may have a more complex role in the cell was provided when they were shown to be identical to the tumor suppressor proteins nm23-H1 and nm23-H2, respectively (7Rosengaard A.M. Krutsch H.C. Shearn A. Biggs J.R. Barker E. Margulies I.M.K. King C.R. Liotta L.A. PubMed Scopus Google Scholar, J.A. A. A.M. King C. 1991; Google Scholar). In in either have been in A. 8: Google Scholar, J. N. J.R. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. I. S. H. H. M. J. PubMed Scopus Google Scholar), the of the of NDK in the cell. role as nucleotide a of have been to these from as histidine kinases to the of as a as a for the a L.A. J. Natl. PubMed Scopus Google and A. PubMed Scopus Google Scholar). of the tumor suppressor has been this to be in cell and R. P. I. C. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a of A. M. Lascu I. J. Lacombe M.-L. PubMed Scopus Google Scholar). the role of NDK is a by the of the of and transfer by the The of the NDK has been in the and a ping-pong of NDK is (4Parks R.E. Agarwal R.P. Enzymes. 1973; 8: 307-334Crossref Scopus (357) Google Scholar, E. 8: PubMed Scopus Google Scholar). the overall is the of a and the NDK, is phosphorylated NDK, and and are and and is the of the the of the ping-pong with a binding site that both of the and both with for the binding is to the state for such a has in different for the to which NDK between different (2Mourad N. Parks R.E. J. Biol. Chem. 1966; 241: 271-278Abstract Full Text PDF PubMed Google Scholar, 3Dumas C. Lascu I. Morera S. Glaser P. Fourme R. Wallet V. Lacombe M.-L. Veron M. Janin J. EMBO J. 1992; 11: 3203-3208Crossref PubMed Scopus (176) Google Scholar, L.A. J. Natl. PubMed Scopus Google Scholar, Agarwal R.P. Parks R.E. PubMed Scopus Google Scholar, M. Veron M. EMBO J. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). has the to such as be in the of the second such the of an of in a of the enzyme to of phosphorylated enzyme and of NDK have been reported of the of a for the was the by J. Biol. Chem. Full Text PDF PubMed Google using and as The Lascu I. M. Veron M. PubMed Scopus Google that the phosphorylation of the active site histidine of NDK from in an of the intrinsic fluorescence that a be We a of the NDK-A and which have been in of phosphoryl transfer was for a range of and analogues. The the of rate and constants for the shown and that for nucleotides the enzyme the nucleotide and followed by rate-limiting phosphoryl transfer. In the enzyme a for adenine and guanine nucleotides with the highest for guanine nucleotides and phosphoryl transfer for adenine nucleotides. and were from or and were from R. S. for by and were to be more than ATPγS and were more than with the nucleotides and at than The NDK-A and -B were a from M.-L. were the J. S. K. C. Full Text PDF PubMed Scopus Google with and an histidine The the site of It the to the of the an For of these were E. of were and was with for at at an of at were by for at in a and by and The cell was by for at in an in a proteins NDK-A and -B were and as S. PubMed Scopus Google Scholar). proteins and cell were and a The was with the and proteins were with in this The were in MOPS, by and as an at were by or by A. of proteins was by in in the of PubMed Scopus Google Scholar). were by the at and using an of PubMed Scopus Google and as are with to was by of μm NDK with an at of or ATPγS for at and a which the nucleotides. The and were for S. M. Lascu I. Janin J. PubMed Scopus Google Scholar). NDK was at in a enzyme (4Parks R.E. Agarwal R.P. Enzymes. 1973; 8: 307-334Crossref Scopus (357) Google using and as in A. The nucleoside diphosphate for NDK was this nucleotide is a for of is as the of of in of The enzyme is for the NDK with and as the is a for was by of was with and of were at and the was by to or by the were on a × with μm on a The was at at an of with J. J. J. A. EMBO J. 1986; PubMed Scopus Google Scholar). rates were from the of and as a of on intrinsic tryptophan fluorescence were on a at The rates of fluorescence were with a or with a and tryptophan fluorescence was at and at to the a of data were using a to in a were with the and to a using a In all enzyme was mixed with at of nucleotide were was to that was in to the of the in the were at in A. and NDK-A and -B were to greater than The proteins NDK-B, and in as at and with the of and respectively. proteins a second at the of the was to of the at the site the than of the for in the enzyme and as at all proteins had of equivalent to rates of of and with and at as by were and s−1, respectively. to of and s−1 before (6Gilles A.M. Presecan E. Vonica A. Lascu I. J. Biol. Chem. 1991; 266: 8784-8789Abstract Full Text PDF PubMed Google for NDK-A and -B with and at active site was by the of with the with μm of the were phosphorylated as reported E. 8: PubMed Scopus Google A. Lascu I. A. Veron M. M. PubMed Scopus Google Scholar). μm was to the intrinsic tryptophan fluorescence by phosphorylated was the of μm in a in The of to or the of to phosphorylated had on the from a small to of the that the observed fluorescence the phosphorylation state of the enzyme and nucleotide binding Lascu I. M. Veron M. PubMed Scopus Google Scholar). In a the tryptophan fluorescence of μm or was at between and μm The change in fluorescence was the concentration and used to the concentration of at which of the fluorescence change when of the is was for the in fluorescence to of the nucleotide by the with of the NDK is and were used to the overall from the The of were μm for and μm for NDK-BHis. was for NDK-B, and was for NDK-BHis. NDK-A a in fluorescence upon the change in fluorescence was upon of than for to this small was for the of to a in this be used to the rate of the phosphorylation a the in fluorescence upon with μm in the in fluorescence was which was by a with an observed rate of change in fluorescence In an μm phosphorylated was mixed with μm and to a in s−1 The of to phosphorylated enzyme change in The was for nucleotide between and μm for and and μm for the was to the of the by the of the these concentration was linearly dependent on nucleotide concentration and The of that the change in fluorescence was by the rate of nucleotide and the of the the second order rate for the nucleotide binding of this second order rate in of the intrinsic rate constants of be with a the from the of the phosphorylated and the is the concentration of is very the a of the concentration of the In this case, are two in which a is for the fluorescence and in which is linearly dependent on concentration. the phosphoryl transfer is faster than nucleotide binding the second order rate is by that the observed is a reaction, and as have a of to s−1, and have little on H. for the Scholar). In the second order rate is of the order for a for as shown but this is of all nucleotides phosphoryl transfer is slower than nucleotide binding is to be hyperbolically dependent on and at such that and the second order rate The in which be this from a change in The data for and and data at the by a of which the two in the of the second order rate constants observed for a range of nucleotides, are too for a The were for and and and the data are in I. proteins have rates of dephosphorylation was 2–3-fold faster for with and for and The of the fluorescence were by the and NDK-A had an than the between the and proteins are the of the have used the more second order rate constants for the observed fluorescence upon with NDK and with phosphorylated NDK (NDK for NDK-B, and order rate NDK in a The in that for the the dephosphorylation is faster than the phosphorylation reaction, to to for all which is in with the data are in the range as for NDK of Lascu I. M. Veron M. PubMed Scopus Google Scholar), for NDK of E. 8: PubMed Scopus Google Scholar), and for NDK of I. H. Presecan E. I. J. PubMed Scopus Google Scholar). The shown in a and were for a range of nucleotides and in case, the be by a The of the nucleotide concentration are shown in The second order rate constants from the of the are in second order rate constants of fluorescence change for and phosphorylated order rate in a For the nucleotides with of and the concentration range was that shown in these In all cases, the were the It is to that for of the nucleotides used the of the from these are different from and have and this that the of a is in these For the of in which both nucleotide binding events are faster than the phosphoryl transfer step, the to the rate be for the and and the is order in both the is is the of a of and the the shown in are order in the but the an of the of the the of is taken from the of the data with NDP, be used to the of the of the by that the The are thus and s−1 for and and they are in with the corresponding nucleotides which be in the of The in that has for nucleotides, with the second order rate constants by more than is consistent with the events is for all nucleotides. of nucleotides with dephosphorylation with or the rates were for guanine and were followed by and cytosine with rates of and respectively. In rates of dephosphorylation were faster for than for for dephosphorylation were faster than such that for the overall was of the with the different nucleotides were with both and NDK-A and of the observed second order rate constants by more than a of from reported for NDK-BHis. the of to different the phosphorylation for a of NTPs on the ribose We and nucleotides phosphorylated the as observed by the in and linearly dependent upon nucleotide concentration. The second order rate constants were × × and × s−1, respectively. In the of in that may be a for The of triphosphate for to with NDK an second order rate of × s−1, which is 13 of the rate with that the group be the binding was observed for and which phosphorylated NDK at 13 of the rate of are in with of J. R. S. C. Lascu I. Janin J. Veron M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google using a nucleotides and are for NDK R.H. PubMed Scopus Google Scholar), and the thiophosphorylated enzyme be and is as as the natural modification of for has been shown to the rate of phosphoryl transfer for a of P.A. Scholar). for natural nucleotides, the fluorescence of on with ATPγS and when the thiophosphorylated enzyme was mixed with the of the fluorescence change was than that observed with In fluorescence be by a but the of were slower than of the equivalent with or and For the of to μm in a in fluorescence with 0.7 the of μm to μm thiophosphorylated a in fluorescence with in to the with nucleotides, the of on nucleotide concentration was but at high nucleotide for both and a and The be by a is or is the of and is the concentration of nucleotide for form of the concentration of is that for a as in the of the group is to the phosphoryl transfer and is to that is this that is In such a case, and for and for transfer to The of the and the second order rate are in for and order rate in a The data in and that the of the second order rate constants for the are of for NDPs, and to as for the natural nucleotides. the between the second order rate constants for the NDPs is for the the order is that the a to by NDK for both of nucleotides. the second order rate is two a different The phosphoryl transfer is faster for adenine nucleotides than for the which were all to a of For adenine and the was slower than dephosphorylation and has a of and for ATPγS and GTPγS, respectively. The more than the equivalent diphosphate the to and for ATPγS and The of guanine nucleotides was and that of adenine was The for and were be they were than was used to NDK-BHis∼thioP, the of change from for from which more the was reduced and the second order rate was reduced with and and and very when dephosphorylation of The nucleoside and GDPβS, were also and be phosphorylated by to form the and The was hyperbolically dependent on of μm and μm and of s−1 and s−1, respectively. transfer of a group from to was by of a of ATPγS, ADPβS, μm and of was to nucleotides The of for of and phosphorylation of are very to more to the than to the thiophosphorylated The phosphoryl transfer was faster for phosphorylation of the two than was of the corresponding with ATPγS and dephosphorylation of thiophosphorylated NDK with and were for NDK-B, that and were of for NDK-BHis. NDK-A was phosphorylated with ATPγS and GTPγS, and for were and and were and s−1, respectively. for NDK-A with were 2–3-fold but the rates were for NDK-A the of GTPγS was than that of in the of NDK-B, GTPγS was more than The for the fluorescence of NDK-A were too small to for dephosphorylation of thiophosphorylated NDK-A with NDK-A and -B have been and from as both and in a that the has on the of the proteins in the the was the to the were all on this We have shown as for the Lascu I. M. Veron M. PubMed Scopus Google Scholar), phosphorylation of the active site histidine in the enzyme in a of the intrinsic which is on NDK-A and -B tryptophan at and H. N. S. M. 1991; PubMed Scopus Google Scholar). The NDK has only a tryptophan at to in and a of fluorescence on phosphorylation Lascu I. M. Veron M. PubMed Scopus Google Scholar). that the fluorescence of the enzyme from the this is in all eukaryotic a fluorescence change may for the may be We used this fluorescence change to the of the enzyme with this of the is a for an enzyme with a ping-pong mechanism. of the data shown are consistent with The observed for all NDK in the were and was linearly dependent on nucleotide concentration the range and μm for the observed is by the of the nucleotide to the for and of the second order rate constants was than that for a is by the with natural and nucleotides in which the varied by more than a of and all nucleotides had a is a binding of nucleotide followed by phosphoryl transfer a change of the which phosphoryl transfer as shown in nucleotide binding is faster than the phosphoryl transfer step, the second order rate constants in and are for phosphorylation and for the and was of the nucleotide used as The only of an NDK was that of NDK by J. Biol. Chem. Full Text PDF PubMed Google using a rates for phosphorylation with μm of s−1 and for dephosphorylation of NDK with μm of are to the rates of s−1 and s−1 from the that the phosphorylation of NDK is in the fluorescence The of the thiophosphate has a on phosphoryl transfer R.H. PubMed Scopus Google Scholar), the of second order rate constants observed for the thiophosphate with the equivalent a change in or with a small change in the of and is by the data for dephosphorylation of NDK by and GDPβS, are to for dethiophosphorylation by and the that and for phosphoryl and transfer the of be are and s−1 for and respectively. be for all of the nucleotides in and that for and is to that of and is s−1 for and that nucleotide binding is to is to the nucleotide rate constants The of and two μm and and for and for and are to be in the range to s−1 and to s−1 for all NDPs. the of binding followed by rate-limiting phosphoryl transfer be for all and In the cases, the rate of nucleotide is the rate of the transfer the rate is s−1, the of binding is for all nucleotides. The that the phosphoryl transfer be too fast to measure by for the with all NDPs. For from the may be too to the to be at a nucleotide concentration. The that the fluorescence for at a of s−1 to this more all were the range of nucleotide for of of at The is to measure of the small but were to data to The data had but a from a to the data of μm and of the data are they are with the from the GTPγS The the rates of the nucleotide step, which occurs before phosphoryl transfer from of in when the the observed to the must be considered as as the with all of ping-pong In case, this is the binding of to NDK and to phosphorylated of the for the but the used by the of ATPγS when phosphorylated NDK with or of when NDK with The data to state that for all and for nucleotides, phosphoryl transfer be For nucleotides, the enzyme has binding of and In the of adenine and the nucleotide may be to the rates. The for the range of nucleotides and in the the is by The second order rate constants were highest with guanine nucleotides, followed by adenine and nucleotides, and with cytosine nucleotides with the rates of and dephosphorylation was as fast as with the nucleotide the the of phosphoryl transfer is the in case, the enzyme the different The between the bases by NDK has been the with between in the and the enzyme which is used to measure only of to be was that NDK is C. Lascu I. Morera S. Glaser P. Fourme R. Wallet V. Lacombe M.-L. Veron M. Janin J. EMBO J. 1992; 11: 3203-3208Crossref PubMed Scopus (176) Google Scholar, S. Lascu I. C. P. Veron M. Janin J. PubMed Scopus Google Scholar, Morera S. Lascu I. Veron M. Janin J. PubMed Scopus Google Scholar), of with different nucleotides for NDK Agarwal R.P. Parks R.E. PubMed Scopus Google Scholar). with nucleotides that the of the on the second order rate constants a from both a change in the of the nucleotide and a change in the phosphoryl transfer was observed that at nucleotide and were the of high but and have the highest The data in that in the have on and but that on are greater with for the of the 2′-hydroxyl on the ribose an in and in a small in for and but a for The of the and the of the nucleotide with NDK have been in of NDK with (NDK of Morera S. Lascu I. Veron M. Janin J. PubMed Scopus Google (NDK of S. Lascu I. C. P. Veron M. Janin J. PubMed Scopus Google NDK of J. S. M. E. J. Biol. PubMed Scopus Google and S. Lacombe M.-L. Janin J. Full Text Full Text PDF PubMed Scopus Google The of the nucleotide is between and and to form a with or via a with and may to guanine from bases S. Lacombe M.-L. Janin J. Full Text Full Text PDF PubMed Scopus Google and to of reported for guanine nucleotides. The ribose and of and form with and S. Lacombe M.-L. Janin J. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the were and a the of 2′-hydroxyl to Morera S. Lascu I. Veron M. Janin J. PubMed Scopus Google Scholar). The of the has a more on the second order rate as shown in this and in J. R. S. C. Lascu I. Janin J. Veron M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). J. R. S. C. Lascu I. Janin J. Veron M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that the with nucleotides is to a of binding but to an of the with the which is to the to be by the of the In have the of the for the rate constants of an enzyme that a ping-pong mechanism. has shown that NDK between different nucleotides both the and the ribose nucleotide be the phosphoryl and and be the phosphoryl may nucleotide to the are consistent with a role of the enzyme as a for maintaining the in the cell. The the of a greater of the and role of NDK in the cell. of the to the NDK and to the of the and transfer by The also a of the of nucleotide analogues such as by NDK and the with which analogues be the cell. is an for the of such analogues as therapeutic J. R. S. C. Lascu I. Janin J. Veron M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. J. I. Biol. PubMed Scopus Google Scholar, A. I. M. J. PubMed Scopus Google Scholar, Morera S. S. R. Veron M. Janin J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We J. and I. Lascu for on the We are to and R. S. for