BACKGROUND
nicotinamide adenine dinucleotide  and nad+ phosphate  are two of the most important coenzymes in cells.
they act as either electron donors or electron acceptors in more than  <dig> enzymatically catalyzed oxidoreductions  <cit> .
nad+ also plays an essential role in producing atp, and is involved in various cellular processes as a substrate for a number of degradation enzymes  <cit> .
abnormal regulation of nad+ metabolism may result in or is associated with serious metabolic disorders and diseases, such as diabetes, cancers, neurological disorders and cardiovascular disease  <cit> .
furthermore, the disruption of nad+ synthesis can cause growth suppression and cell death  <cit> .
nad+ can be synthesized de novo from simple amino acid precursors such as tryptophan or aspartate, or converted from intermediates such as nicotinamide , nicotinic acid  or nicotinamide riboside  via salvage pathways, which are designated as salvage pathways i  na → namn  → denad  → nad+), ii , and iii , respectively   <cit> .
all three pathways are in fact interconnected.
however, some organisms  may lack a nicotinamidase  to prevent nam from entering pathway i, whereas others  lack a nicotinamide phosphoribosyl transferase  to prevent nam from entering pathway ii <cit> .
in yeast, pathway i may be extended by first converting nr to nam  <cit> .
some nad+-consuming enzymes may break down nad+ to form various types of adp-ribosyl groups, in which the nam moiety is the most common end-product  <cit> .
in a variety of physiological events, some of these enzymes  can be significantly activated, such as during the regulation of apoptosis, dna replication, and dna repair  <cit> , thus potentially leading to the rapid depletion of intracellular nad+, and associated accumulation of nam  <cit> .
since nam is also known as a strong inhibitor of several nad+-consuming enzymes, uncontrolled nam accumulation may negatively affect not only nad+ metabolism, but also cellular functions such as gene silencing, hst1-mediated transcriptional repression, and life span of cells  <cit> .
therefore, nad+ salvage pathways i and ii are important not only in regenerating nad+, but also in preventing the accumulation of nam.
while nam may be converted to nmn by nmprt  or na by pnca , there were no reports on other enzymes that might act on nam.
in the present study, we have discovered by genetic and biochemical approaches that xanthosine phosphorylase  is also capable of converting nam to nr in e. coli.
xapa was originally identified from e. coli, and known to catalyze the reversible ribosyltransfer on purine nucleosides including xanthosine, inosine and guanosine  <cit> .
our data has not only assigned a novel function to xapa, but also uncovered a potential new route in the nad+ salvage, in which the pathway iii is extended by using nam as an alternative precursor in xapa-possessing organisms.
RESULTS
genetic disruption of nad+de novo biosynthesis and nad+ salvage pathway i in escherichia coli
in an effort to uncover the new function of e. coli xapa in nad+ salvage pathway from nicotinamide, we produced a set of gene knockout mutants deficient in previously defined nad+ synthetic pathways, including nad+de novo and nad+ salvage pathways i and iii for genetic investigation purpose .
we first generated a mutant strain deficient in nad+de novo pathway  that was unable to survive in the m <dig> minimal medium, but could restore the growth to a level comparable to the wild-type bw <dig> when na or nam was supplied to allow nad+ synthesized via nad+ salvage pathway i .
escherichia coli
strains and plasmids used in this study
+
*cgsc is the e. coli genetic stock center of yale university.
escherichia coli
strains in different culture media*
+
*notes: na, nicotinic acid; nam, nicotinamide; nad+, nicotinamide adenine dinucleotide; nt, not tested; –, no proliferation; +, proliferation; dn, de novo nad+ synthesis; i, nad+ salvage pathway i; iii, nad+ salvage pathway iii.
we then generated double-deletion mutant bw25113ΔnadcΔpnca to also interrupt the conversion from nam to na in nad+ salvage pathway i. this mutant was expected to only survive in the absence of na, but not nam due to the lack of nad+ salvage pathway ii in e. coli .
the growth of bw25113ΔnadcΔpnca mutant in the absence of na was confirmed as expected, but we also unexpectedly observed its survival in m9/nam medium, albeit with a much slower growth rate  .
this result suggested the presence of another unknown salvage pathway can participate in the conversion of nam from medium into nad+.
genetic evidence on the involvement of xapa in nad+ salvage pathway
the ability for bw25113ΔnadcΔpnca to grow in m9/nam medium implied a previously undefined enzyme might be involved in feeding nam into the nad+ synthesis.
the poor efficiency in utilizing nam was indicative of the presence of an enzyme that might use nam as an atypical substrate, but the activity was sufficient for bacterial growth when other nad+ intermediates were unavailable.
based on the substrate preference of xapa towards purine nucleosides and the fact that its sister enzyme deod  is able to use nr as a non-typical substrate to form nam in vitro <cit> , we hypothesized that xapa might be a candidate enzyme responsible for converting nam to nr.
to test this hypothesis, we developed three multiple gene deletion mutants, namely, bw25113ΔnadcΔpncaΔxapa, bw25113ΔnadcΔpncaΔnadr, and bw25113ΔnadcΔpncaΔxapaΔnadr .
among them, the growth of bw25113ΔnadcΔpncaΔxapa was worse than that of bw25113ΔnadcΔpnca in the m9/nam medium  .
when a complementary plasmid pbad-xapa  was reintroduced into this triple-deletion mutant, its growth rate was restored to a similar level of that of bw25113ΔnadcΔpnca .
we also assessed the growth of this triple-deletion strain in m9/nad+ medium, and observed its normal growth in a dose-dependent manner .
the potential involvement of other unknown pathway in making nad+ could be ruled out, since this triple-deletion transformed with pbad-xapa was unable to growth in the m <dig> minimal medium .
the contribution of xapa in nad+ salvaging was further tested by generating mutants with additional deletion of nadr .
both mutants were able to grow in m9/na medium, but not in m <dig> or m9/nam medium , indicating that nr produced by xapa from nam was connected to the nadr-mediated nad+ salvage pathway iii.
collectively, these observations implied the capability for xapa to use nam as a less efficient substrate to produce nr that could be routed into the pathway iii  in vivo.
biochemical evidence on the conversion of nr from nam by e. coli xapa
the genetic data on the involvement of xapa in converting nam to nr was further validated by biochemical assays using recombinant xapa protein that was expressed using an e. coli expression system and purified into homogeneity .
standard nr sample used in these assays was prepared by a hydrolysis of 5′-phosphate groups from nmn by ciap.
the ability for xapa to convert nam to nr was first confirmed by hplc-esi-ms/ms assay.
in reactions catalyzed by recombinant xapa and ciap , selected-ion monitoring chromatogram  detected a single peak at the retention time corresponding to nr .
further positive ms/ms analysis at m/z  <dig> detected two major peaks with m/z at  <dig> and  <dig>  representing nr  and the nam  moiety, respectively , which confirmed the xapa-catalyzed production of nr from nam.
further kinetic analysis showed that the
k
m
value towards nam was  <dig>  mm, and the
v
max
was at  <dig> nmol/min/mg protein.
the kinetic data indicated that xapa in e. coli was much less efficient in using nam to synthesize nr than using typical substrate   <cit> , or when compared with other nad+ salvaging enzymes   <cit> , but similar to those of deod  from calf and e. coli  in converting the non-typical substrate nr to nam  <cit> .
the contribution of xapa in nad+ salvaging was also confirmed in bacterial mutants cultured in m9/nam medium, in which the consumption of extracellular nam by the triple-deletion  was reduced by 95% in comparison to that by the double-deletion bw25113ΔnadcΔpnca .
the consumption of extracellular nam was restored when vector expressing xapa  was reintroduced to the triple-deletion .
the level of intracellular nad+ was detectable in bw25113ΔnadcΔpnca , but virtually undetectable in bw25113ΔnadcΔpncaΔxapa .
again, the intracellular nad+ level could be restored by reintroducing xapa into the triple-deletion, but not by egfp .
discussion
contribution of xapa to an alternative nad+ salvage pathway from nam
xanthosine phosphorylase  is a second purine nucleoside phosphorylase  in e. coli.
similar to pnp-i , it mainly functions in the purine metabolism by carrying out both phosphorylation and synthesis of purine and purine deoxy-/ribonucleosides  <cit> .
here we first obtained genetic evidence that xapa was probably involved in nad+ salvage in e. coli.
we also provided more direct biochemical evidences that xapa was able to synthesize nr from nam.
both bacterial growth experiments and enzyme kinetic data indicated that xapa used nam in a much less efficient way than using its typical substrates , suggesting that nam served only as a non-typical substrate, which was comparable to the pnp-i.
therefore, the capability to convert nam to nr appeared to be a “side effect” for xapa.
however, such a side-effect was sufficient to maintain the survival of e. coli by feeding nam into the salvage pathway iii when all other nad+ synthetic pathways were unavailable and only nam was present in the minimal medium.
for clarity, we designated the xapa-mediated extension of nad+ salvage pathway iii as the salvage pathway iiib  .
potential uses of xapa-mediated salvage pathway in drug development
the true biological function of pathway iiib may be less significant in e. coli, as this bacterium is able to synthesize nad+ via multiple routes .
however, we speculate that it may be highly significant for some other pathogenic bacteria that lack nad+de novo, nad+ salvage pathway i and/or ii for nad+ synthesis.
one of the examples might be the gram-negative coccobacillus pasteurella multocida that causes a range of diseases in humans and animals.
it appears to be v-factor-independent, indicating its capability to utilize nam as the pyridine nucleotide, as well as nad+, nmn and nr to synthesize nad+ <cit> .
analysis of nad+ biosynthesis pathways reveals that p. multocida lacks nad+de novo and nad+ salvage pathway i but possesses nad+ salvage pathway ii and nad+ salvage pathway iii for the presence of nadv, nmprt homolog in bacteria, and nadr  <cit>  .
furthermore, a pnp homologue  is also present in the p. multocida genome.
accordingly, it seems reasonable to speculate that p. multocida may synthesize nad+ from nam through nad+ salvage pathway ii and/or nad+ salvage pathway iiib.
however, the hypothesis on the potential contribution of nad+ salvage pathway iiib to nad+ biosynthesis in such bacteria remains to be tested.
if the hypothesis is confirmed, the xapa or its isoenzyme may be explored as a novel target for developing therapeutics.
in fact, the nad+ salvage pathways of human is similar to that of p. multocida in that humans lack nad+ salvage pathway i, but possess nmprt-mediated nad+ salvage pathway ii and nrk -mediated nad+ salvage pathway iii   <cit> .
nmprt is highly expressed in many types of tumor cells, including human hematologic malignancies, to maintain adequate levels of nad+ <cit> .
inhibitor of nmprt, such as fk <dig>  has been in phase ii clinical trials  <cit> .
however, nam was found to have an antidote potential for the cellular effects of fk <dig>  <cit> , which indicates that the nad+ synthesis pathways from nam may be not completely disrupted.
as the pnp-mediated new salvage pathway is also present in mammals , it remains to be tested whether human pnp  is also able to utilize nam to synthesize nr as an alternative to pathway ii , thus responsible for the slow anti-cancer action of fk <dig>  in fact, the enzymes involved in the pathway iiib, such as human pnp and nrk, are all effective anticancer drug targets  <cit> .
when the inhibitors of pnp and/or nrk are used in combination with fk <dig>  rapid nad+ depletion and nam accumulation may occur, hopefully increase the anticancer efficacy or widen the antitumor spectra, or even conquer the drug resistance.
contribution of xapa to a new pyridine nucleotide cycle
additionally, this newly discovered pathway iiib may also be significant in the pyridine nucleotide cycles  that are mediated by the breakdown and re-synthesis of nad+ <cit> .
pncs are economic and efficient approaches to recycle nad+ intermediates back into nad+ without the actual consumption of nad+, which ensures the homeostatic balance between nad+ degradation and replenishment.
thus far, pncs are found to consist of three to seven reaction steps, which are correspondingly named as pnc iii–vii   <cit> .
when nad+ is broken down to nam by the nad+-consuming enzymes, the nam-based nad+ re-synthetic pathways involved in pncs are identical to the nad+ salvage pathways.
more specifically, the salvage pathway i and ii are the same as the nad+ resynthesis routes of pnc v and pnc iii, respectively.
therefore, the presence of xapa-mediated nad+ salvage pathway iiib would also extend the related pnc iv, which is proposed here as pnc iv-b to distinguish it from the existing pcn-iv cycle .
CONCLUSIONS
we have provided genetic and biochemical evidences showing that xanthosine phosphorylase  in e. coli is able to utilize nicotinamide  as an atypical substrate to synthesize nicotinamide riboside , which extends the nad+ salvage pathway iii to use nr as an alternative precursor .
this unexpected discovery not only assigns a new function to xapa, but also increases our current knowledge on the nad+ biosynthesis and pyridine nucleotide cycles.

 nicotinamide adenine dinucleotide and nad+ phosphate are two of the most important coenzymes in cells. they act as either electron donors or electron acceptors in more than enzymatically catalyzed oxidoreductions. nad+ also plays an essential role in producing atp, and is involved in various cellular processes as a substrate for a number of degradation enzymes. abnormal regulation of nad+ metabolism may result in or is associated with serious metabolic disorders and diseases, such as diabetes, cancers, neurological disorders and cardiovascular disease. furthermore, the disruption of nad+ synthesis can cause growth suppression and cell death. nad+ can be synthesized de novo from simple amino acid precursors such as tryptophan or aspartate, or converted from intermediates such as nicotinamide, nicotinic acid or nicotinamide riboside via salvage pathways, which are designated as salvage pathways i na → namn → denad → nad+), ii, and iii, respectively. all three pathways are in fact interconnected. however, some organisms may lack a nicotinamidase to prevent nam from entering pathway i, whereas others lack a nicotinamide phosphoribosyl transferase to prevent nam from entering pathway ii. in yeast, pathway i may be extended by first converting nr to nam. some nad+-consuming enzymes may break down nad+ to form various types of adp-ribosyl groups, in which the nam moiety is the most common end-product. in a variety of physiological events, some of these enzymes can be significantly activated, such as during the regulation of apoptosis, dna replication, and dna repair, thus potentially leading to the rapid depletion of intracellular nad+, and associated accumulation of nam. since nam is also known as a strong inhibitor of several nad+-consuming enzymes, uncontrolled nam accumulation may negatively affect not only nad+ metabolism, but also cellular functions such as gene silencing, hst1-mediated transcriptional repression, and life span of cells. therefore, nad+ salvage pathways i and ii are important not only in regenerating nad+, but also in preventing the accumulation of nam. while nam may be converted to nmn by nmprt or na by pnca, there were no reports on other enzymes that might act on nam. in the present study, we have discovered by genetic and biochemical approaches that xanthosine phosphorylase is also capable of converting nam to nr in e. coli. xapa was originally identified from e. coli, and known to catalyze the reversible ribosyltransfer on purine nucleosides including xanthosine, inosine and guanosine. our data has not only assigned a novel function to xapa, but also uncovered a potential new route in the nad+ salvage, in which the pathway iii is extended by using nam as an alternative precursor in xapa-possessing organisms.  genetic disruption of nad+de novo biosynthesis and nad+ salvage pathway i in escherichia coli in an effort to uncover the new function of e. coli xapa in nad+ salvage pathway from nicotinamide, we produced a set of gene knockout mutants deficient in previously defined nad+ synthetic pathways, including nad+de novo and nad+ salvage pathways i and iii for genetic investigation purpose. we first generated a mutant strain deficient in nad+de novo pathway that was unable to survive in the m minimal medium, but could restore the growth to a level comparable to the wild-type bw when na or nam was supplied to allow nad+ synthesized via nad+ salvage pathway i. escherichia coli strains and plasmids used in this study + *cgsc is the e. coli genetic stock center of yale university. escherichia coli strains in different culture media* + *notes: na, nicotinic acid; nam, nicotinamide; nad+, nicotinamide adenine dinucleotide; nt, not tested; –, no proliferation; +, proliferation; dn, de novo nad+ synthesis; i, nad+ salvage pathway i; iii, nad+ salvage pathway iii. we then generated double-deletion mutant bw25113ΔnadcΔpnca to also interrupt the conversion from nam to na in nad+ salvage pathway i. this mutant was expected to only survive in the absence of na, but not nam due to the lack of nad+ salvage pathway ii in e. coli. the growth of bw25113ΔnadcΔpnca mutant in the absence of na was confirmed as expected, but we also unexpectedly observed its survival in m9/nam medium, albeit with a much slower growth rate. this result suggested the presence of another unknown salvage pathway can participate in the conversion of nam from medium into nad+. genetic evidence on the involvement of xapa in nad+ salvage pathway the ability for bw25113ΔnadcΔpnca to grow in m9/nam medium implied a previously undefined enzyme might be involved in feeding nam into the nad+ synthesis. the poor efficiency in utilizing nam was indicative of the presence of an enzyme that might use nam as an atypical substrate, but the activity was sufficient for bacterial growth when other nad+ intermediates were unavailable. based on the substrate preference of xapa towards purine nucleosides and the fact that its sister enzyme deod is able to use nr as a non-typical substrate to form nam in vitro, we hypothesized that xapa might be a candidate enzyme responsible for converting nam to nr. to test this hypothesis, we developed three multiple gene deletion mutants, namely, bw25113ΔnadcΔpncaΔxapa, bw25113ΔnadcΔpncaΔnadr, and bw25113ΔnadcΔpncaΔxapaΔnadr. among them, the growth of bw25113ΔnadcΔpncaΔxapa was worse than that of bw25113ΔnadcΔpnca in the m9/nam medium. when a complementary plasmid pbad-xapa was reintroduced into this triple-deletion mutant, its growth rate was restored to a similar level of that of bw25113ΔnadcΔpnca. we also assessed the growth of this triple-deletion strain in m9/nad+ medium, and observed its normal growth in a dose-dependent manner. the potential involvement of other unknown pathway in making nad+ could be ruled out, since this triple-deletion transformed with pbad-xapa was unable to growth in the m minimal medium. the contribution of xapa in nad+ salvaging was further tested by generating mutants with additional deletion of nadr. both mutants were able to grow in m9/na medium, but not in m or m9/nam medium, indicating that nr produced by xapa from nam was connected to the nadr-mediated nad+ salvage pathway iii. collectively, these observations implied the capability for xapa to use nam as a less efficient substrate to produce nr that could be routed into the pathway iii in vivo. biochemical evidence on the conversion of nr from nam by e. coli xapa the genetic data on the involvement of xapa in converting nam to nr was further validated by biochemical assays using recombinant xapa protein that was expressed using an e. coli expression system and purified into homogeneity. standard nr sample used in these assays was prepared by a hydrolysis of 5′-phosphate groups from nmn by ciap. the ability for xapa to convert nam to nr was first confirmed by hplc-esi-ms/ms assay. in reactions catalyzed by recombinant xapa and ciap, selected-ion monitoring chromatogram detected a single peak at the retention time corresponding to nr. further positive ms/ms analysis at m/z detected two major peaks with m/z at and representing nr and the nam moiety, respectively, which confirmed the xapa-catalyzed production of nr from nam. further kinetic analysis showed that the k m value towards nam was mm, and the v max was at nmol/min/mg protein. the kinetic data indicated that xapa in e. coli was much less efficient in using nam to synthesize nr than using typical substrate, or when compared with other nad+ salvaging enzymes, but similar to those of deod from calf and e. coli in converting the non-typical substrate nr to nam. the contribution of xapa in nad+ salvaging was also confirmed in bacterial mutants cultured in m9/nam medium, in which the consumption of extracellular nam by the triple-deletion was reduced by 95% in comparison to that by the double-deletion bw25113ΔnadcΔpnca. the consumption of extracellular nam was restored when vector expressing xapa was reintroduced to the triple-deletion. the level of intracellular nad+ was detectable in bw25113ΔnadcΔpnca, but virtually undetectable in bw25113ΔnadcΔpncaΔxapa. again, the intracellular nad+ level could be restored by reintroducing xapa into the triple-deletion, but not by egfp. discussion contribution of xapa to an alternative nad+ salvage pathway from nam xanthosine phosphorylase is a second purine nucleoside phosphorylase in e. coli. similar to pnp-i, it mainly functions in the purine metabolism by carrying out both phosphorylation and synthesis of purine and purine deoxy-/ribonucleosides. here we first obtained genetic evidence that xapa was probably involved in nad+ salvage in e. coli. we also provided more direct biochemical evidences that xapa was able to synthesize nr from nam. both bacterial growth experiments and enzyme kinetic data indicated that xapa used nam in a much less efficient way than using its typical substrates, suggesting that nam served only as a non-typical substrate, which was comparable to the pnp-i. therefore, the capability to convert nam to nr appeared to be a “side effect” for xapa. however, such a side-effect was sufficient to maintain the survival of e. coli by feeding nam into the salvage pathway iii when all other nad+ synthetic pathways were unavailable and only nam was present in the minimal medium. for clarity, we designated the xapa-mediated extension of nad+ salvage pathway iii as the salvage pathway iiib. potential uses of xapa-mediated salvage pathway in drug development the true biological function of pathway iiib may be less significant in e. coli, as this bacterium is able to synthesize nad+ via multiple routes. however, we speculate that it may be highly significant for some other pathogenic bacteria that lack nad+de novo, nad+ salvage pathway i and/or ii for nad+ synthesis. one of the examples might be the gram-negative coccobacillus pasteurella multocida that causes a range of diseases in humans and animals. it appears to be v-factor-independent, indicating its capability to utilize nam as the pyridine nucleotide, as well as nad+, nmn and nr to synthesize nad+. analysis of nad+ biosynthesis pathways reveals that p. multocida lacks nad+de novo and nad+ salvage pathway i but possesses nad+ salvage pathway ii and nad+ salvage pathway iii for the presence of nadv, nmprt homolog in bacteria, and nadr. furthermore, a pnp homologue is also present in the p. multocida genome. accordingly, it seems reasonable to speculate that p. multocida may synthesize nad+ from nam through nad+ salvage pathway ii and/or nad+ salvage pathway iiib. however, the hypothesis on the potential contribution of nad+ salvage pathway iiib to nad+ biosynthesis in such bacteria remains to be tested. if the hypothesis is confirmed, the xapa or its isoenzyme may be explored as a novel target for developing therapeutics. in fact, the nad+ salvage pathways of human is similar to that of p. multocida in that humans lack nad+ salvage pathway i, but possess nmprt-mediated nad+ salvage pathway ii and nrk -mediated nad+ salvage pathway iii. nmprt is highly expressed in many types of tumor cells, including human hematologic malignancies, to maintain adequate levels of nad+. inhibitor of nmprt, such as fk has been in phase ii clinical trials. however, nam was found to have an antidote potential for the cellular effects of fk, which indicates that the nad+ synthesis pathways from nam may be not completely disrupted. as the pnp-mediated new salvage pathway is also present in mammals, it remains to be tested whether human pnp is also able to utilize nam to synthesize nr as an alternative to pathway ii, thus responsible for the slow anti-cancer action of fk in fact, the enzymes involved in the pathway iiib, such as human pnp and nrk, are all effective anticancer drug targets. when the inhibitors of pnp and/or nrk are used in combination with fk rapid nad+ depletion and nam accumulation may occur, hopefully increase the anticancer efficacy or widen the antitumor spectra, or even conquer the drug resistance. contribution of xapa to a new pyridine nucleotide cycle additionally, this newly discovered pathway iiib may also be significant in the pyridine nucleotide cycles that are mediated by the breakdown and re-synthesis of nad+. pncs are economic and efficient approaches to recycle nad+ intermediates back into nad+ without the actual consumption of nad+, which ensures the homeostatic balance between nad+ degradation and replenishment. thus far, pncs are found to consist of three to seven reaction steps, which are correspondingly named as pnc iii–vii. when nad+ is broken down to nam by the nad+-consuming enzymes, the nam-based nad+ re-synthetic pathways involved in pncs are identical to the nad+ salvage pathways. more specifically, the salvage pathway i and ii are the same as the nad+ resynthesis routes of pnc v and pnc iii, respectively. therefore, the presence of xapa-mediated nad+ salvage pathway iiib would also extend the related pnc iv, which is proposed here as pnc iv-b to distinguish it from the existing pcn-iv cycle.  we have provided genetic and biochemical evidences showing that xanthosine phosphorylase in e. coli is able to utilize nicotinamide as an atypical substrate to synthesize nicotinamide riboside, which extends the nad+ salvage pathway iii to use nr as an alternative precursor. this unexpected discovery not only assigns a new function to xapa, but also increases our current knowledge on the nad+ biosynthesis and pyridine nucleotide cycles. 
in an effort to reconstitute the nad+ synthetic pathway in escherichia coli, we produced a set of gene knockout mutants with deficiencies in previously well-defined nad+de novo and salvage pathways. unexpectedly, the mutant deficient in nad+de novo and salvage pathway i could grow in m9/nicotinamide medium, which was contradictory to the proposed classic nad+ metabolism of e. coli. we speculate that this xapa-mediated nad+ salvage pathway might be significant in some bacteria lacking nad+de novo and nad+ salvage pathway i or ii, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize nad+. such e. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the nad+ biosynthesis. we wanted to verify whether xanthosine phophorylase contributed to a new nad+ salvage pathway from nicotinamide. both the genetic and biochemical evidences indicated that xapa could convert nicotinamide to nicotinamide riboside in e. coli, albeit with relatively weak activity, indicating that xapa may contribute to a second nad+ salvage pathway from nicotinamide. to further validate the new function of xapa, we cloned and expressed e. coli xapa as a recombinant soluble protein. additional knockout of xapa further slowed down the bacterial growth in m9/nicotinamide medium, whereas the complementation of xapa restored the growth phenotype. biochemical assay confirmed that xapa was capable of using nicotinamide as a substrate for nicotinamide riboside formation. purine nucleoside phosphorylasenicotinamide ribosidesalvage pathwaypyridine nucleotide cycles
 in an effort to reconstitute the nad+ synthetic pathway in escherichia coli, we produced a set of gene knockout mutants with deficiencies in previously well-defined nad+de novo and salvage pathways. unexpectedly, the mutant deficient in nad+de novo and salvage pathway i could grow in m9/nicotinamide medium, which was contradictory to the proposed classic nad+ metabolism of e. coli. such e. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the nad+ biosynthesis. we wanted to verify whether xanthosine phophorylase contributed to a new nad+ salvage pathway from nicotinamide.  additional knockout of xapa further slowed down the bacterial growth in m9/nicotinamide medium, whereas the complementation of xapa restored the growth phenotype. to further validate the new function of xapa, we cloned and expressed e. coli xapa as a recombinant soluble protein. biochemical assay confirmed that xapa was capable of using nicotinamide as a substrate for nicotinamide riboside formation.  both the genetic and biochemical evidences indicated that xapa could convert nicotinamide to nicotinamide riboside in e. coli, albeit with relatively weak activity, indicating that xapa may contribute to a second nad+ salvage pathway from nicotinamide. we speculate that this xapa-mediated nad+ salvage pathway might be significant in some bacteria lacking nad+de novo and nad+ salvage pathway i or ii, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize nad+. however, this speculation needs to be experimentally tested. purine nucleoside phosphorylasenicotinamide ribosidesalvage pathwaypyridine nucleotide cycles 