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MetaCyc Enzyme: xanthosine phosphorylase

Gene: xapA Accession Numbers: G85 (MetaCyc), b2407, ECK2401

Synonyms: pndA, purine nucleoside phosphorylase II

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of xanthosine phosphorylase = [XapA]6
         xanthosine phosphorylase = XapA

Summary:
Xanthosine phosphorylase (XapA or PNP-II) is one of two purine nucleoside phosphorylases (PNPs) in E. coli. PNPs use orthophosphate to cleave the N-glycosidic bond of the β-(deoxy)ribonucleosides to produce α-(deoxy)ribose1-phosphate and the free purine base. The bases can be utilized as precursors in the synthesis of nucleotides in the purine salvage pathways, as well as a nitrogen source. The pentose-1-phosphate formed serves as a carbon source. The other PNP, PNP-I purine nucleoside phosphorylase, differs in substrate specificity.

XapA does not cleave or synthesize adenosine and deoxyadenosine but catalyzes the phosphorolysis of xanthosine, inosine and guanosine with comparable efficiency. Therefore, this enzyme has been variously referred to as xanthosine phosphorylase and inosine-guanosine phosphorylase [Koszalka88, Dandanell05]. XapA enables E. coli to grow on xanthosine as the sole source of carbon [HammerJespersen80, Seeger95]. The amino acid sequence of XapA resembles that of mammalian purine nucleoside phosphorylases and has no similarity with PNP-I [Dandanell05].

Recently, an investigation of the NAD+ salvage pathway showed that a ΔnadC pncA double mutant can grow slowly in M9 minimal medium containing nicotinamide (NAM). This implied the presence of a previously undefined activity that routes NAM into NAD+synthesis. An additional mutation in xapA further decreased growth of the ΔnadC pncA mutant on NAM, and purified XapA was shown to catalyze formation of nicotinamide riboside from nicotinamide at low efficiency [Dong14].

XapA predominantly exists as a hexamer with some evidence for co-existence of a trimeric from [Dandanell05, HammerJespersen80]. Crystal structures of XapA were solved in the presence of guanine and phosphate as well as xanthine and sulfate. XapA forms a hexamer in a dimer-of-trimers conformation in the crystal structure [Dandanell05]. A Y191L mutation in the active site resulted in no detectable activity of XapA against xanthosine, although it retained activity against inosine and guanosine with altered affinities and reduced maximal velocities, while an N239D mutation changed the substrate specificity to adenosine [Dandanell05].

A genetic engineering study was directed at inosine production using a xapA mutant. It was concluded that due to apparent regulatory features in E. coli, other engineered organisms show higher inosine production [Shimaoka06].

XapA from E. coli K-12 can also catalyze the degradation of the synthetic xenobiotic substrate 5-nitrobenzisoxazole by the Kemp elimination mechanism, which is not known to occur in biological systems. This property has been demonstrated in enzyme engineering studies [Khersonsky11].

The XapR-xanthosine DNA-binding transcriptional activator and the presence of the inducer xanthosine in the growth medium are required for full expression of XapA [HammerJespersen80, Jorgensen99]. Regulatory mutants were first isolated as second-site revertants of a deoD mutant that were able to catabolize purine ribonucleosides [Buxton80]. A xapA mutant can no longer grow on xanthosine as the sole source of carbon [Seeger95].

Xap: "xanthosine phosphorylase" [Buxton80]

Review: [Kulikowska04]

Citations: [Bezirdzhian86, Kocharian86, Bezirdzhian87, Bezirdzhian87a, Konrad12]

Locations: cytosol

Map Position: [2,522,067 <- 2,522,900]

Molecular Weight of Polypeptide: 29.835 kD (from nucleotide sequence), 30.1 kD (experimental) [Seeger95 ]

Molecular Weight of Multimer: 205.0 kD (experimental) [HammerJespersen80]

Unification Links: ASAP:ABE-0007927 , CGSC:9 , EchoBASE:EB4152 , EcoGene:EG20250 , EcoliWiki:b2407 , Entrez-gene:946878 , ModBase:P45563 , OU-Microarray:b2407 , PortEco:xapA , PR:PRO_000024228 , Protein Model Portal:P45563 , RefSeq:NP_416902 , RegulonDB:G85 , SMR:P45563 , String:511145.b2407 , UniProt:P45563

Relationship Links: InterPro:IN-FAMILY:IPR000845 , InterPro:IN-FAMILY:IPR001369 , InterPro:IN-FAMILY:IPR010943 , InterPro:IN-FAMILY:IPR011268 , InterPro:IN-FAMILY:IPR018099 , Panther:IN-FAMILY:PTHR11904 , Panther:IN-FAMILY:PTHR11904:SF9 , PDB:Structure:1YQQ , PDB:Structure:1YQU , PDB:Structure:1YR3 , Pfam:IN-FAMILY:PF01048 , Prosite:IN-FAMILY:PS01240

Gene-Reaction Schematic: ?

Instance reactions of [a purine ribonucleoside + phosphate ↔ a purine base + α-D-ribose-1-phosphate] (2.4.2.1):
i1: adenosine + phosphate ↔ adenine + α-D-ribose-1-phosphate (2.4.2.1)

i2: guanosine + phosphate ↔ guanine + α-D-ribose-1-phosphate (2.4.2.1/2.4.2.15)

i3: inosine + phosphate ↔ hypoxanthine + α-D-ribose-1-phosphate (2.4.2.1)

GO Terms:

Biological Process: GO:0006148 - inosine catabolic process Inferred from experiment [Koszalka88, Shimaoka06, Dandanell05]
GO:0006149 - deoxyinosine catabolic process Inferred from experiment [Koszalka88]
GO:0006152 - purine nucleoside catabolic process Inferred from experiment [Seeger95, Koszalka88, Shimaoka06, Dandanell05, Jorgensen99]
GO:0006161 - deoxyguanosine catabolic process Inferred from experiment [Koszalka88]
GO:0015949 - nucleobase-containing small molecule interconversion Inferred from experiment [Seeger95]
GO:0034214 - protein hexamerization Inferred from experiment [Dandanell05]
GO:0034355 - NAD salvage Inferred from experiment [Dong14]
GO:0046115 - guanosine catabolic process Inferred from experiment [Koszalka88, Dandanell05]
GO:0055086 - nucleobase-containing small molecule metabolic process Inferred from experiment Inferred by computational analysis [GOA01, Seeger95]
GO:1903228 - xanthosine catabolic process Inferred from experiment [Seeger95]
GO:0006139 - nucleobase-containing compound metabolic process Inferred by computational analysis [GOA01]
GO:0009116 - nucleoside metabolic process Inferred by computational analysis [GOA01]
Molecular Function: GO:0004731 - purine-nucleoside phosphorylase activity Inferred from experiment Inferred by computational analysis [GOA01a, GOA01, Seeger95, Koszalka88, Shimaoka06, Jorgensen99, Dandanell05]
GO:0042802 - identical protein binding Inferred from experiment [HammerJespersen80]
GO:0047724 - inosine nucleosidase activity Inferred from experiment [Koszalka88, Shimaoka06, Dandanell05]
GO:0047975 - guanosine phosphorylase activity Inferred from experiment [Koszalka88, Dandanell05]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016757 - transferase activity, transferring glycosyl groups Inferred by computational analysis [UniProtGOA11]
GO:0016763 - transferase activity, transferring pentosyl groups Inferred by computational analysis [GOA01]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment Inferred by computational analysis [GOA01, Dandanell05]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: metabolism central intermediary metabolism nucleotide and nucleoside conversions

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: xanthosine phosphorylase

Synonyms: purine nucleoside phosphorylase

xanthosine + phosphate <=> α-D-ribose-1-phosphate + xanthine

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

In Pathways: purine nucleotides degradation II (aerobic) , xanthine and xanthosine salvage , urate biosynthesis/inosine 5'-phosphate degradation , purine ribonucleosides degradation

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Kinetic Parameters:

Substrate
Km (μM)
Vmax (µmol mg-1 min-1)
Citations
xanthosine
72.0
8.7
[Dandanell05]
phosphate
760.0
[Koszalka88]

pH(opt) (forward direction): 5.8-7.2 [Dandanell05]

pH(opt) (reverse direction): 5.5-8 [Dandanell05]


Enzymatic reaction of: inosine phosphorylase (xanthosine phosphorylase)

Synonyms: purine nucleoside phosphorylase

inosine + phosphate <=> hypoxanthine + α-D-ribose-1-phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for inosine: 2'-deoxyinosine [HammerJespersen80 ]

Alternative Products for α-D-ribose-1-phosphate: deoxyribose 1-phosphate [Koszalka88 ]

In Pathways: purine nucleotides degradation II (aerobic) , adenine and adenosine salvage III , adenosine nucleotides degradation II , purine ribonucleosides degradation

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
Apparent activation energies in the phosphorolytic and synthetic direction are 11.5 Kcal/mol and 7.9 Kcal/mol respectively [Koszalka88].

The enzyme can catalyze the reverse reaction, synthesis of inosine, in vitro [HammerJespersen80, Koszalka88].

Kinetic Parameters:

Substrate
Km (μM)
Vmax (µmol mg-1 min-1)
Citations
inosine
963.0
11.9
[Dandanell05]
phosphate
760.0
[Koszalka88]
α-D-ribose-1-phosphate
59.0
[Koszalka88]
hypoxanthine
3.3
[Koszalka88]
deoxyribose 1-phosphate
58.0
[Koszalka88]
2'-deoxyinosine
62.0
[Koszalka88]

pH(opt) (forward direction): 6.6 [Koszalka88]

pH(opt) (reverse direction): 6.8 [Koszalka88]


Enzymatic reaction of: guanosine phosphorylase (xanthosine phosphorylase)

Synonyms: purine nucleoside phosphorylase

EC Number: 2.4.2.15

guanosine + phosphate <=> guanine + α-D-ribose-1-phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for guanosine: 2'-deoxyguanosine [Buxton80 ]

In Pathways: purine nucleotides degradation II (aerobic) , superpathway of guanine and guanosine salvage , guanosine nucleotides degradation III , guanine and guanosine salvage , purine ribonucleosides degradation

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Kinetic Parameters:

Substrate
Km (μM)
Vmax (µmol mg-1 min-1)
Citations
guanine
4.1
[Koszalka88]
phosphate
760.0
[Koszalka88]
guanosine
155.0
14.2
[Dandanell05]
α-D-ribose-1-phosphate
59.0
[Koszalka88]
2'-deoxyguanosine
44.0
[Koszalka88]

pH(opt) (forward direction): 6.5-7.5 [Dandanell05]

pH(opt) (reverse direction): 6.5-8 [Dandanell05]


Enzymatic reaction of: nicotinamide riboside phosphorylase (xanthosine phosphorylase)

EC Number: 2.4.2.-

nicotinamide + α-D-ribose-1-phosphate + H+ <=> 1-(β-D ribofuranosyl)nicotinamide + phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

Reversibility of this reaction is unspecified.

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Kinetic Parameters:

Substrate
Km (μM)
Vmax (µmol mg-1 min-1)
Citations
nicotinamide
5810.0
[Dong14]
nicotinamide
0.4


Sequence Features

Feature Class Location Citations Comment
Amino-Acid-Sites-That-Bind 65
[UniProt12b]
UniProt: Substrate 2; via tele nitrogen.
Protein-Segment 85 -> 87
[UniProt12b]
UniProt: Substrate 2 binding; Sequence Annotation Type: region of interest.
Amino-Acid-Sites-That-Bind 117
[UniProt12b]
UniProt: Substrate 2; via amide nitrogen.
Sequence-Conflict 144
[Seeger95, UniProt12b]
Alternate sequence: A; UniProt: (in Ref. 1; CAA52049).
Mutagenesis-Variant 191
[Dandanell05, UniProt12b]
Alternate sequence: L; UniProt: No detectable activity with xanthosine as substrate, but largely retains its activity against other substrates, namely inosine and guanosine, although with altered affinities, higher and lower respectively, and clearly reduced maximal velocities for both.
Amino-Acid-Sites-That-Bind 197
[UniProt12b]
UniProt: Substrate 1.
Amino-Acid-Sites-That-Bind 216
[UniProt12b]
UniProt: Substrate 2.
Mutagenesis-Variant 239
[Dandanell05, UniProt12b]
Alternate sequence: D; UniProt: Catalyzes the phosphorolysis of adenosine with moderate efficiency, and essentially has lost all activity against the 6-oxo-purine substrates xanthosine, inosine and guanosine.
Amino-Acid-Sites-That-Bind 239
[UniProt12b]
UniProt: Substrate 1.
Sequence-Conflict 261
[Seeger95, UniProt12b]
Alternate sequence: H; UniProt: (in Ref. 1; CAA52049).
Sequence-Conflict 265
[Seeger95, UniProt12b]
Alternate sequence: L; UniProt: (in Ref. 1; CAA52049).

History:
10/20/97 Gene b2407 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene G85.


References

Bezirdzhian86: Bezirdzhian KhO, Kocharian ShM, Akopian ZhI (1986). "[Isolation of the hexameric form of purine nucleoside phosphorylase from E. coli. Comparative study of trimeric and hexameric forms of the enzyme]." Biokhimiia 1986;51(7);1085-92. PMID: 3089333

Bezirdzhian87: Bezirdzhian KhO, Kocharian ShM, Akopian ZhI (1987). "[Hexameric purine nucleoside phosphorylase II from Escherichia coli K-12. Physico-chemical and catalytic properties and stabilization with substrates]." Biokhimiia 1987;52(10);1624-31. PMID: 3122852

Bezirdzhian87a: Bezirdzhian KhO, Kocharian ShM, Akopian ZhI (1987). "[Hexamere purine nucleoside phosphorylase from Escherichia coli K-12. Kinetic analysis and mechanism of reaction]." Biokhimiia 52(11);1770-6. PMID: 3125860

Buxton80: Buxton RS, Hammer-Jespersen K, Valentin-Hansen P (1980). "A second purine nucleoside phosphorylase in Escherichia coli K-12. I. Xanthosine phosphorylase regulatory mutants isolated as secondary-site revertants of a deoD mutant." Mol Gen Genet 179(2);331-40. PMID: 7007808

Dandanell05: Dandanell G, Szczepanowski RH, Kierdaszuk B, Shugar D, Bochtler M (2005). "Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene." J Mol Biol 348(1);113-25. PMID: 15808857

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Dong14: Dong WR, Sun CC, Zhu G, Hu SH, Xiang LX, Shao JZ (2014). "New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD(+) salvage from nicotinamide." BMC Microbiol 14;29. PMID: 24506841

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

HammerJespersen80: Hammer-Jespersen K, Buxton RS, Hansen TD (1980). "A second purine nucleoside phosphorylase in Escherichia coli K-12. II. Properties of xanthosine phosphorylase and its induction by xanthosine." Mol Gen Genet 1980;179(2);341-8. PMID: 7007809

Jorgensen99: Jorgensen C, Dandanell G (1999). "Isolation and characterization of mutations in the Escherichia coli regulatory protein XapR." J Bacteriol 181(14);4397-403. PMID: 10400599

Khersonsky11: Khersonsky O, Malitsky S, Rogachev I, Tawfik DS (2011). "Role of chemistry versus substrate binding in recruiting promiscuous enzyme functions." Biochemistry 50(13);2683-90. PMID: 21332126

Kocharian86: Kocharian ShM, Melkumian MA (1986). "[Genetic analysis of Escherichia coli K-12 mutants defective for the structural and regulatory genes for second purine nucleoside phosphorylase]." Genetika 22(8);2055-65. PMID: 3095187

Konrad12: Konrad A, Piškur J, Liberles DA (2012). "The evolution of catalytic residues and enzyme mechanism within the bacterial nucleoside phosphorylase superfamily 1." Gene 510(2);154-61. PMID: 22967797

Koszalka88: Koszalka GW, Vanhooke J, Short SA, Hall WW (1988). "Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12." J Bacteriol 170(8);3493-8. PMID: 3042752

Kulikowska04: Kulikowska E, Kierdaszuk B, Shugar D (2004). "Xanthine, xanthosine and its nucleotides: solution structures of neutral and ionic forms, and relevance to substrate properties in various enzyme systems and metabolic pathways." Acta Biochim Pol 51(2);493-531. PMID: 15218545

Seeger95: Seeger C, Poulsen C, Dandanell G (1995). "Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli." J Bacteriol 177(19);5506-16. PMID: 7559336

Shimaoka06: Shimaoka M, Takenaka Y, Mihara Y, Kurahashi O, Kawasaki H, Matsui H (2006). "Effects of xapA and guaA disruption on inosine accumulation in Escherichia coli." Biosci Biotechnol Biochem 70(12);3069-72. PMID: 17151449

Tomisek65: Tomisek AJ, Hoskins AP, Reid MR (1965). "Chromatographic studies of purine metabolism. VI. Inhibition of inosine phosphorylase by 6-mercaptopurine." Cancer Res 25(11);1925-32. PMID: 5323535

UniProt12b: UniProt Consortium (2012). "UniProt version 2012-11 released on 2012-11-26 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Mon Nov 24, 2014, BIOCYC14B.