Guvermectin Biosynthesis Revealing the Key Role of a Phosphoribohydrolase and Structural Insight into the Active Glutamate of a Noncanonical Adenine Phosphoribosyltransferase.

Guvermectin is a novel plant growth regulator that has been registered as a new agrochemical in China. It is an adenosine analogue with an unusual psicofuranose instead of ribose. Herein, the gene cluster responsible for guvermectin biosynthesis in Streptomyces caniferus NEAU6 is identified using gene interruption and heterologous expression experiments. A key intermediate psicofuranine 6'-phosphate (PMP) is chemically synthesized, and the functions of GvmB, C, D, and E are verified by individual stepwise enzyme reactions in vitro. The results also show that the biosynthesis of guvermectin is coupled with adenosine production by a single cluster. The higher catalytic efficiency of GvmB on PMP than AMP ensures the effective biosynthesis of guvermectin. Moreover, a phosphoribohydrolase GvmA is employed in the pathway that can hydrolyze AMP but not PMP and shows higher catalytic efficiency for the AMP hydrolysis than that of the AMP dephosphorylation by GvmB, leading to shunting of adenosine biosynthesis toward the production of guvermectin. Finally, the crystal structure of GvmE in complex with the product PMP has been solved. Glu160 at the C-terminal is identified as the acid/base for protonation/deprotonation of N7 of the adenine ring, demonstrating that GvmE is a noncanonical adenine phosphoribosyltransferase.

How a purine salvage enzyme singles out the right base.

Two phosphoribosyltransferases in the purine salvage pathway exhibit exquisite substrate specificity despite the chemical similarity of their distinct substrates, but the basis for this discrimination was not fully understood. Ozeir et al. now employ a complementary biochemical, structural, and computational approach to deduce the chemical constraints governing binding and propose a distinct mechanism for catalysis in one of these enzymes, adenine phosphoribosyltransferase. These insights, built on data from an unexpected finding, finally provide direct answers to key questions regarding these enzymes and substrate recognition more generally.

Structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed reaction.

The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.

Crystal structures of APRT from Francisella tularensis - an N-H···N hydrogen bond imparts adenine specificity in adenine phosporibosyltransferases.

Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine. ENZYME: EC 2.4.2.7 DATABASE: Structural data are available in Protein Data Bank (PDB) under the accession numbers 5YW2 and 5YW5.

Structural Insights into the Forward and Reverse Enzymatic Reactions in Human Adenine Phosphoribosyltransferase.

Phosphoribosyltransferases catalyze the displacement of a PRPP α-1'-pyrophosphate to a nitrogen-containing nucleobase. How they control the balance of substrates/products binding and activities is poorly understood. Here, we investigated the human adenine phosphoribosyltransferase (hAPRT) that produces AMP in the purine salvage pathway. We show that a single oxygen atom from the Tyr105 side chain is responsible for selecting the active conformation of the 12 amino acid long catalytic loop. Using in vitro, cellular, and in crystallo approaches, we demonstrated that Tyr105 is key for the fine-tuning of the kinetic activity efficiencies of the forward and reverse reactions. Together, our results reveal an evolutionary pressure on the strictly conserved Tyr105 and on the dynamic motion of the flexible loop in phosphoribosyltransferases that is essential for purine biosynthesis in cells. These data also provide the framework for designing novel adenine derivatives that could modulate, through hAPRT, diseases-involved cellular pathways.

Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase.

Phosphoribosyl transferases (PRTs) are essential in nucleotide synthesis and salvage, amino acid, and vitamin synthesis. Transition state analysis of several PRTs has demonstrated ribocation-like transition states with a partial positive charge residing on the pentose ring. Core chemistry for synthesis of transition state analogues related to the 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) reactant of these enzymes could be developed by stereospecific placement of bis-phosphate groups on an iminoaltritol ring. Cationic character is provided by the imino group and the bis-phosphates anchor both the 1- and 5-phosphate binding sites. We provide a facile synthetic path to these molecules. Cyclic-nitrone redox methodology was applied to the stereocontrolled synthesis of three stereoisomers of a selectively monoprotected diol relevant to the synthesis of transition-state analogue inhibitors. These polyhydroxylated pyrrolidine natural product analogues were bis-phosphorylated to generate analogues of the ribocationic form of 5-phosphoribosyl 1-phosphate. A safe, high yielding synthesis of the key intermediate represents a new route to these transition state mimics. An enantiomeric pair of iminoaltritol bis-phosphates (L-DIAB and D-DIAB) was prepared and shown to display inhibition of Plasmodium falciparum orotate phosphoribosyltransferase and Saccharomyces cerevisiae adenine phosphoribosyltransferase (ScAPRT). Crystallographic inhibitor binding analysis of L- and D-DIAB bound to the catalytic sites of ScAPRT demonstrates accommodation of both enantiomers by altered ring geometry and bis-phosphate catalytic site contacts.

The biosynthetic pathway of 2-azahypoxanthine in fairy-ring forming fungus.

"Fairy rings" resulting from fungus-stimulated plant growth occur all over the world. In 2010, 2-azahypoxanthine (AHX) from a fungus Lepista sordida was identified as the "fairy" that stimulates plant growth. Furthermore, 2-aza-8-oxohypoxanthine (AOH) was isolated as a common metabolite of AHX in plants, and the endogenous existence of AHX and AOH in plants was proved. The structure of AHX allowed us to hypothesize that AHX was derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Thus, we performed a feeding experiment that supplied AICAR to L. sordida. Consumption of AICAR and accumulation of AHX were observed after feeding. The mycelia extract had enzymatic activity of adenine/5-aminoimidazole-4-carboxamide phosphoribosyltransferase (APRT). APRT gene of L. sordida revealed its structural characteristics in homology modeling and showed transcriptional enhancement after feeding. These results support that AHX was synthesized from AICAR and AHX biosynthesis was transcriptionally controlled by AICAR, indicating the presence of novel purine metabolic pathway in L. sordida.

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded ß-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and ß-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

Adenine phosphoribosyltransferase (APRT) deficiency: identification of a novel nonsense mutation.

BACKGROUND: Adenine phosphoribosyltransferase deficiency (APRTD) is an under estimated genetic form of kidney stones and/or kidney failure, characterized by intratubular precipitation of 2,8-dihydroxyadenine crystals (2,8-DHA). Currently, five pathologic allelic variants have been identified as responsible of the complete inactivation of APRT protein. CASE PRESENTATION: In this study, we report a novel nonsense mutation of the APRT gene from a 47- year old Italian patient. The mutation, localized in the exon 5, leads to the replacement of a cytosine with a thymine (g.2098C > T), introducing a stop codon at amino acid position 147 (p.Gln147X).This early termination was deleterious for the enzyme structural and functional integrity, as demonstrated by the structure analysis and the activity assay of the mutant APRT protein. CONCLUSION: These data revealed that the p.Gln147X mutation in APRT gene might be a new cause of APRT disease.

Identification and characterization of a novel adenine phosphoribosyltransferase gene (ZmAPT2) from maize (Zea mays L.).

Adenine phosphoribosyltransferase (APRT) is the key enzyme that converts adenine to adenosine monophosphate (AMP) in the purine salvage pathway. It was found that several different forms of APRT gene exist in plants, but no APRT gene in maize has been reported up to now. In this study, a novel maize APRT gene was cloned and characterized through a combination of bioinformatic, RT-PCR and RACE strategies. The full length of APRT cDNA sequence is 1202 nucleotides, with an ORF encoding 214 amino acid residues. Alignment of the deduced protein with that of other plant APRT genes indicates that the new gene is the form 2 of maize APRT, thus it was named ZmAPT2. Through basic local alignment search tool, search in the genomic survey sequence database of MaizeGDB, the putative genomic sequence of ZmAPT2 was obtained. Comparison of the cDNA and genomic sequence of the ZmAPT2 gene revealed that it contained seven exons and six introns. The locations of the introns within the maize ZmAPT2 coding region were consistent with those in the previously isolated APRTs of arabidopsis and rice. RT-PCR analysis showed that ZmAPRT was constitutively expressing in different organs under high temperature and salt stresses. Southern blot analysis indicated that at least three APRT genes existed in maize genome. These results confirmed that the novel maize ZmAPT2 gene was truly identified, and its potential role in maize growth and development was discussed.

Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism.

Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 A resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 A resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 A resolution. Comparisons of these three hAPRT structures with other 'type I' PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPP. Comparative analyses presented here provide structural evidence to propose the role of Glu104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism.

[Bioinformatic identification and analysis of Schistosoma japonicum adenine phosphoribosyltransferase].

OBJECTIVE: To identify adenine phosphoribosyltransferases in Schistosoma japonicum and analyze their structural features. METHODS: Based on the accessible transcriptome and proteomic data, the S. japonicum adenine phosphoribosyl transferases were identified using bioinformatics approaches including bi-directional homology comparison, domain search and phylogenetic analysis. Homology modeling was also performed to describe the structural features of the proteins. RESULTS AND CONCLUSION: Two homologue sequences of adenine phosphoribosyltransferase were obtained from S. japonicum, and the EST abundance, physico-chemical properties and three-dimensional structures of them were also acquired.

Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells.

Gene targeting via homologous recombination is a powerful tool for studying gene function, but the targeting efficiency in human cell lines is too low for generating knockout mutants. Several cell lines null for the gene responsible for Bloom syndrome, BLM, have shown elevated targeting efficiencies. Therefore, we reasoned that gene targeting would be enhanced by transient suppression of BLM expression by RNA interference. To test this, we constructed a gene correction assay system to measure gene targeting frequencies using a disrupted hypoxanthine phosphoribosyltransferase (HPRT) locus in the human HT1080 cell line, and examined the effect of small interfering RNA (siRNA) for BLM on gene targeting. When HPRT-null cells pretreated with BLM siRNA were co-transfected with the siRNA and a gene correction vector, the gene targeting frequency was elevated three-fold, while the random integration frequency was marginally affected. Remarkably, in BLM heterozygous (+/-) cells derived from HPRT-null cells, the BLM siRNA treatment gave more than five-fold higher targeting frequencies, even with one-tenth the amount of BLM siRNA used for BLM+/+ cells. Furthermore, in the human pre-B cell line Nalm-6, the siRNA treatment enhanced gene targeting 6.3-fold and > 5.8-fold at the HPRT and adenine phosphoribosyltransferase (APRT) loci, respectively. These results indicate that transient suppression of BLM expression by siRNA stimulates gene targeting in human cells, facilitating a further improvement of gene targeting protocols for human cell lines.

Cloning and characterization of a second form of the rice adenine phosphoribosyl transferase gene (OsAPT2) and its association with TGMS.

A rice gene, OsAPT2, which encodes a putative adenine phosphoribosyl transferase (APRT), was cloned and characterized. Analysis of the cDNA and genomic sequences revealed seven exons and six introns in the OsAPT2. The deduced amino acid sequence of OsAPT2 is highly homologous to those of previously isolated APRTs. RT-PCR analysis indicated that the OsAPT2 transcript in the young panicles of 'Annong S-1' is down-regulated at 29 degrees C, the critical temperature for induction of 'Annong S-1' fertility conversion. Since the panicle is likely the thermo-sensitive organ at the early stages of pollen fertility alternation, the observed heat-induced change in the OsAPT2 expression pattern in young panicles may mediate, at least in part, thermo-sensitive genic male sterility (TGMS) in 'Annong S-1'. An antisense strategy was used to suppress the expression of the OsAPT2 homolog in Arabidopsis, and the obtained homozygous transgenic plants contained lower AMP content, displayed lower pollen germination rates and exhibited some abnormalities in leaf phenotypes and flowering timing. These data suggest that OsAPT2 is likely to be involved in TGMS in the rice line 'Annong S-1'.

Three-dimensional structure of human adenine phosphoribosyltransferase and its relation to DHA-urolithiasis.

In mammals, adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) is present in all tissues and provides the only known mechanism for the metabolic salvage of adenine resulting from the polyamine biosynthesis pathway or from dietary sources. In humans, APRT deficiency results in serious kidney illness such as nephrolithiasis, interstitial nephritis, and chronic renal failure as a result of 2,8-dihydroxyadenine (DHA) precipitation in the renal interstitium. To address the molecular basis of DHA-urolithiasis, the recombinant human APRT was crystallized in complex with adenosine 5'-monophosphate (AMP). Refinement of X-ray diffraction data extended to 2.1 A resolution led to a final crystallographic R(factor) of 13.3% and an R(free) of 17.6%. This structure is composed of nine beta-strands and six alpha-helices, and the active site pocket opens slightly to accommodate the AMP product. The core of APRT is similar to that of other phosphoribosyltransferases (PRTases), although the adenine-binding domain is quite different. Structural comparisons between the human APRT and other "type I" PRTases of known structure revealed several important features of the biochemistry of PRTases. We propose that the residues located at positions corresponding to Leu159 and Ala131 in hAPRT are responsible for the base specificities of type I PRTases. The comparative analysis shown here also provides structural information for the mechanism by which mutations in the human APRT lead to DHA-urolithiasis.

Crystal structure of adenine phosphoribosyltransferase from Leishmania tarentolae: potential implications for APRT catalytic mechanism.

The three-dimensional structure of Leishmania tarentolae adenine phosphoribosyltransferase (APRT) in complex with adenosine-5-monophosphate (AMP) and a phosphate ion has been solved. Refinement against X-ray diffraction data extending to 2.2-A resolution led to a final crystallographic R factor of 18.3%. Structural comparisons amongst this APRT enzyme and other 'type I' PRTases whose structures have been determined reveal several important features of the PRTases catalytic mechanism. Based on structural superpositions and molecular interaction potential calculations, it was possible to suggest that the PRPP is the first substrate to bind, while the AMP is the last product to leave the active site, in accordance to recent kinetic studies performed with the Leishmania donovani APRT.

Closed site complexes of adenine phosphoribosyltransferase from Giardia lamblia reveal a mechanism of ribosyl migration.

The adenine phosphoribosyltransferase (APRTase) from Giardia lamblia was co-crystallized with 9-deazaadenine and sulfate or with 9-deazaadenine and Mg-phosphoribosylpyrophosphate. The complexes were solved and refined to 1.85 and 1.95 A resolution. Giardia APRTase is a symmetric homodimer with the monomers built around Rossman fold cores, an element common to all known purine phosphoribosyltransferases. The catalytic sites are capped with a small hood domain that is unique to the APRTases. These structures reveal several features relevant to the catalytic function of APRTase: 1) a non-proline cis peptide bond (Glu(61)-Ser(62)) is required to form the pyrophosphate binding site in the APRTase.9dA.MgPRPP complex but is a trans peptide bond in the absence of pyrophosphate group, as observed in the APRTase.9dA.SO4 complex; 2) a catalytic site loop is closed and fully ordered in both complexes, with Glu(100) from the catalytic loop acting as the acid/base for protonation/deprotonation of N-7 of the adenine ring; 3) the pyrophosphoryl charge is neutralized by a single Mg2+ ion and Arg(63), in contrast to the hypoxanthine-guanine phosphoribosyltransferases, which use two Mg2+ ions; and 4) the nearest structural neighbors to APRTases are the orotate phosphoribosyltransferases, suggesting different paths of evolution for adenine relative to other purine PRTases. An overlap comparison of AMP and 9-deazaadenine plus Mg-PRPP at the catalytic sites of APRTases indicated that reaction coordinate motion involves a 2.1-A excursion of the ribosyl anomeric carbon, whereas the adenine ring and the 5-phosphoryl group remained fixed. G. lamblia APRTase therefore provides another example of nucleophilic displacement by electrophile migration.

The adenine phosphoribosyltransferase from Giardia lamblia has a unique reaction mechanism and unusual substrate binding properties.

Purine phosphoribosyltransferases catalyze the Mg2+ -dependent reaction that transforms a purine base into its corresponding nucleotide. They are present in a wide variety of organisms including plants, mammals, and parasitic protozoa. Giardia lamblia, the causative agent of giardiasis, lacks de novo purine biosynthesis and relies primarily on adenine and guanine phosphoribosyltransferases (APRTase and GPRTase) constituting two independent and essential purine salvage pathways. The APRTase from G. lamblia was cloned and expressed with a 6-His tag at its C terminus and purified to apparent homogeneity. Adenine and alpha-d-5-phosphoribosyl-1-pyrophosphate (PRPP) have K(m) values of 4.2 and 143 microm with a k(cat) of 2.8 s(-1) in the forward reaction, whereas AMP and PP(i) have K(m) values of 87 and 450 microm with a k(cat) of 9.5 x 10(-3) s(-1) in the reverse reaction. Product inhibition studies indicated that the forward reaction follows a random Bi Bi mechanism. Results from the kinetics of equilibrium isotope exchange further verified a random Bi Bi mechanism in the forward reaction. In a mutant enzyme, F25W, with kinetic constants similar to those of the wild type and a tryptophan residue at the adenine binding site, the addition of adenine or AMP to the free mutant enzyme resulted in fluorescence quenching, whereas PRPP caused fluorescence enhancement. The dissociation constants thus estimated are 16.5 microm for adenine, 14.3 microm for AMP, and 83.0 microm for PRPP. PP(i) exerted no detectable effect on the tryptophan fluorescence at all, suggesting a lack of PP(i) binding to the free enzyme. An ordered substrate binding in the reverse reaction with AMP bound first followed by PP(i) is thus postulated.

Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae.

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop.