Adenine phosphoribosyltransferase (APRTase) is anenzyme encoded by theAPRTgene, found inhumans onchromosome 16.[5] It is part of the Type I PRTase family and is involved in thenucleotide salvage pathway, which provides an alternative tonucleotide biosynthesis de novo in humans and most other animals.[6] In parasiticprotozoa such asgiardia, APRTase provides the sole mechanism by which AMP can be produced.[7] APRTase deficiency contributes to the formation of kidney stones (urolithiasis) and to potentialkidney failure.[8]
The APRT gene is constituted by 5 exons (in blue). The start (ATG) and stop (TGA) codons are indicated (bold blue). CpG dinucleotides are emphasized in red. They are more abundant in the upstream region of the gene where they form aCpG island.
ARPTase catalyzes a phosphoribosyl transfer from PRPP to adenine, forming AMP and releasing pyrophosphate (PPi).
In organisms that can synthesizepurines de novo, the nucleotide salvage pathway provides an alternative that is energetically more efficient. It can salvage adenine from thepolyamine biosynthetic pathway or from dietary sources of purines.[6] Although APRTase is functionally redundant in these organisms, it becomes more important during periods of rapid growth, such as embryogenesis and tumor growth.[9] It is constitutively expressed in all mammalian tissue.[10]
Inprotozoan parasites, the nucleotide salvage pathway provides the sole means for nucleotide synthesis. Since the consequences of APRTase deficiency in humans is comparatively mild and treatable, it may be possible to treat certainparasitic infections by targeting APRTase function.[11]
APRTase is ahomodimer, with 179amino acid residues permonomer. Each monomer contains the following regions:
Catalytic site of APRTase with reactants adenine and PRPP resolved. The Hood is believed to be important for purine specificity, while the flexible loop is thought to contain the molecules within the active site.
"Core" domain (residues 33-169) with five parallelβ-sheets
"Hood" domain (residues 5-34) with 2α-helices and 2 β-sheets
"Flexible loop" domain (residues 95-113) with 2 antiparallel β-sheets[10]
Residues A131, L159, V25, and R27 are important for purine specificity in human APRTase.
The core is highly conserved across many PRTases. The hood, which contains theadeninebinding site, has more variability within the family of enzymes. A 13-residue motif comprises thePRPP binding region and involves two adjacentacidic residues and at least one surroundinghydrophobic residue.[13]
The enzyme's specificity for adenine involves hydrophobic residuesAla131 andLeu159 in the core domain. In humans, two residues in the hood domainhydrogen bond with the purine for further specificity:Val25 with thehydrogens on N6, andArg27 with N1. Although the flexible loop does not interact with the hood during purine recognition, it is thought to close over theactive site and sequester the reaction fromsolvents.[10]
Most research on APRTase reports that Mg2+ is essential for phosphoribosyl transfer, and this is conserved across Type I PRTases.[12] However, a recent effort to resolve the structure of human APRTase was unable to locate a single site for Mg2+, but did find evidence to suggest a Cl− atom near Trp98. Despite the difficulty of placing Mg2+, it is generally accepted that thecatalytic mechanism is dependent on this ion.[6]
APRTase proceeds via a bi bi ordered sequential mechanism, involving the formation of a ternary complex. The enzyme first bindsPRPP, followed byadenine. After the phosphoribosyl transfer occurs,pyrophosphate leaves first, followed byAMP. Kinetic studies indicate that the phosphoribosyl transfer is relatively fast, while the product release (particularly the release of AMP) israte-limiting.[9]
In human APRTase, it is thought that adenine's N9 proton is abstracted byGlu104 to form an oxacarbeniumtransition state. This functions as thenucleophile to attack theanomeric carbon of PRPP, forming AMP and displacing pyrophosphate from PRPP. The mechanism of APRTase is generally consistent with that of other PRTases, which conserve the function of displacing PRPP's α-1-pyrophosphate using anitrogen nucleophile, in either an SN1 or SN2 attack.[6]
ARPTase deficiency was first diagnosed in theUK in 1976. Since then, two categories of APRTase deficiency have been defined in humans.[14]
Type I deficiency results in a complete loss of APRTase activity and can occur in patients that arehomozygous orcompound heterozygous for variousmutations.[15]Sequencing has revealed many different mutations that can account for Type 1, includingmissense mutations,nonsense mutations, a duplicated set of 4base pairs inexon 3,[16] and a singlethymineinsertion inintron 4.[17] These mutations cause effects that are clustered into three main areas: in the binding of PRPP's β-phosphate, in the binding of PRPP's 5'-phosphate, and in the segment of the flexible loop that closes over the active site during catalysis[10] Type I deficiency has been observed in various ethnic groups but studied predominately amongWhite populations.[17]
Type II deficiency causes APRTase to have a reduced affinity for PRPP, resulting in a tenfold increase in the KM value.[6] It has been observed and studied primarily inJapan.[17]
A diagnosis of APRTase deficiency can be made by analyzingkidney stones, measuring DHA concentrations in urine, or analyzing APRTase activity inerythrocytes. It is treatable with regular doses ofallopurinol orfebuxostat, which inhibit xanthine dehydrogenase activity to prevent the accumulation and precipitation of DHA.[18] The condition can also be attenuated with a low-purine diet and high fluid intake.[14]
^abcdeSilva CH, Silva M, Iulek J, Thiemann OH (Jun 2008). "Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism".Journal of Biomolecular Structure & Dynamics.25 (6):589–97.doi:10.1080/07391102.2008.10507205.PMID18399692.S2CID40788077.
^abShi W, Tanaka KS, Crother TR, Taylor MW, Almo SC, Schramm VL (Sep 2001). "Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae".Biochemistry.40 (36):10800–9.doi:10.1021/bi010465h.PMID11535055.
^abBashor C, Denu JM, Brennan RG, Ullman B (Mar 2002). "Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani".Biochemistry.41 (12):4020–31.doi:10.1021/bi0158730.PMID11900545.
^abcdSilva M, Silva CH, Iulek J, Thiemann OH (Jun 2004). "Three-dimensional structure of human adenine phosphoribosyltransferase and its relation to DHA-urolithiasis".Biochemistry.43 (24):7663–71.doi:10.1021/bi0360758.PMID15196008.
Tischfield JA, Engle SJ, Gupta PK, Bye S, Boyadjiev S, Shao C, O'Neill P, Albertini RJ, Stambrook PJ, Sahota AS (1995). "Germline and Somatic Mutation at the APRT Locus of Mice and Man".Purine and Pyrimidine Metabolism in Man VIII. Advances in Experimental Medicine and Biology. Vol. 370. pp. 661–4.doi:10.1007/978-1-4615-2584-4_137.ISBN978-1-4613-6105-3.PMID7660991.
Takeuchi H, Kaneko Y, Fujita J, Yoshida O (Apr 1993). "A case of a compound heterozygote for adenine phosphoribosyltransferase deficiency (APRT*J/APRT*Q0) leading to 2,8-dihydroxyadenine urolithiasis: review of the reported cases with 2,8-dihydroxyadenine stones in Japan".The Journal of Urology.149 (4):824–6.doi:10.1016/s0022-5347(17)36222-5.PMID8455250.
Ludwig H, Kuzmits R, Pietschmann H, Müller MM (Nov 1979). "Enzymes of the purine interconversion system in chronic lymphatic leukemia: decreased purine nucleoside phosphorylase and adenosine deaminase activity".Blut.39 (5):309–15.doi:10.1007/BF01014193.PMID116697.S2CID6283377.
Johnson LA, Gordon RB, Emmerson BT (Apr 1977). "Adenine phosphoribosyltransferase: a simple spectrophotometric assay and the incidence of mutation in the normal population".Biochemical Genetics.15 (3–4):265–72.doi:10.1007/BF00484458.PMID869896.S2CID41264715.
Chen J, Sahota A, Stambrook PJ, Tischfield JA (Jul 1991). "Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase".Mutation Research.249 (1):169–76.Bibcode:1991MRFMM.249..169C.doi:10.1016/0027-5107(91)90143-C.PMID2067530.
Gathof BS, Sahota A, Gresser U, Chen J, Stambrook PJ, Tischfield JA, Zöllner N (Dec 1990). "Identification of a splice mutation at the adenine phosphoribosyltransferase locus in a German family".Klinische Wochenschrift.69 (24):1152–5.doi:10.1007/BF01815434.PMID2135300.S2CID11791868.
Chen J, Sahota A, Martin GF, Hakoda M, Kamatani N, Stambrook PJ, Tischfield JA (Jun 1993). "Analysis of germline and in vivo somatic mutations in the human adenine phosphoribosyltransferase gene: mutational hot spots at the intron 4 splice donor site and at codon 87".Mutation Research.287 (2):217–25.Bibcode:1993MRFMM.287..217C.doi:10.1016/0027-5107(93)90014-7.PMID7685481.
Sahota A, Chen J, Boyadjiev SA, Gault MH, Tischfield JA (May 1994). "Missense mutation in the adenine phosphoribosyltransferase gene causing 2,8-dihydroxyadenine urolithiasis".Human Molecular Genetics.3 (5):817–8.doi:10.1093/hmg/3.5.817.PMID7915931.
