Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases

Home , Thymidine phosphorylase, Uridine, Uridine phosphorylase

Review Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases

Irina A. Il’icheva †, Konstantin M. Polyakov † and Sergey N. Mikhailov * Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str. 32, 119991 Moscow, Russia; [email protected] (I.A.I.); [email protected] (K.M.P.) * Correspondence: [email protected]; Tel.: +7-499-135-9733 Both authors contributed equally. † Received: 28 February 2020; Accepted: 1 April 2020; Published: 5 April 2020

Abstract: Nucleoside phosphorylases catalyze the reversible phosphorolysis of nucleosides to heterocyclic bases, giving α-d-ribose-1-phosphate or α-d-2-deoxyribose-1-phosphate. These enzymes are involved in salvage pathways of nucleoside biosynthesis. The level of these enzymes is often elevated in tumors, which can be used as a marker for cancer diagnosis. This review presents the analysis of conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases of the first (NP-1) family, which includes hexameric and trimeric purine nucleoside phosphorylases (EC 2.4.2.1), hexameric and trimeric 50-deoxy-50-methylthioadenosine phosphorylases (EC 2.4.2.28), and uridine phosphorylases (EC 2.4.2.3). Nucleosides adopt similar conformations in complexes, with these conformations being significantly different from those of free nucleosides. In complexes, pentofuranose rings of all nucleosides are at the W region of the pseudorotation cycle that corresponds to the energy barrier to the N S interconversion. In most of the complexes, ↔ the orientation of the bases with respect to the ribose is in the high-syn region in the immediate vicinity of the barrier to syn anti transitions. Such conformations of nucleosides in complexes ↔ are unfavorable when compared to free nucleosides and they are stabilized by interactions with the enzyme. The sulfate (or phosphate) ion in the active site of the complexes influences the conformation of the furanose ring. The binding of nucleosides in strained conformations is a characteristic feature of the enzyme–substrate complex formation for this enzyme group.

Keywords: nucleoside phosphorylases; X-ray structures of nucleoside phosphorylases; nucleoside conformations

1. Introduction

1.1. Nucleoside Phosphorylases as Key Enzymes in Nucleoside Metabolism Nucleoside phosphorylases (NPs) catalyze the phosphorolysis of the N-glycosidic bond in purine or pyrimidine β-d-ribo-(2’-deoxyribo)nucleosides and are found in almost all organisms. This class of enzymes includes purine nucleoside phosphorylases (PNPs; EC 2.4.2.1), 50-deoxy-50-methylthioadenosine phosphorylases (MTAPs; EC 2.4.2.28), uridine phosphorylases (UPs; EC 2.4.2.3), pyrimidine nucleoside phosphorylases (PyNPs; EC 2.4.2.2), and thymidine phosphorylases (TPs; EC 2.4.2.4). Scheme1 depicts the general scheme of phosphorolysis of nucleosides by NPs. The equilibrium of the reaction is shifted towards nucleosides, the shift being much more signiﬁcant for PNPs as compared with UPs. Uridine phosphorylases catalyze phosphorolysis of both thymidine and uridine [1]. Purine nucleoside phosphorylases cleave the N-glycosidic bond in purine ribonucleosides and 2’-deoxynucleosides.

Biomolecules 2020, 10, 552; doi:10.3390/biom10040552 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 552 2 of 14 Biomolecules 2020, 10, x FOR PEER REVIEW 2 of 15

SchemeScheme 1. Phosphorolysis 1. Phosphorolysis of nucleosides of nucleosides catalyzed catalyzed by nucleoside by nucleoside phosphorylases phosphorylases (NPs). (NPs Urd—uridine,). Urd— UP—uridineuridine, UP phosphorylase,—uridine phosphorylase, PNP—purine PNP nucleoside—purine nucleoside phosphorylase, phosphorylase, Rib-1-P— Ribα-d-1-ribose-1-phosphate-P—α-D-ribose- , Pi—inorganic1-phosphate, phosphate Pi—inorganic ion. phosphate ion.

ReactionsReactions that that are are catalyzed catalyzed by by nucleoside nucleoside phosphorylasesphosphorylases play play an an important important role role in maintaining in maintaining nucleosidenucleoside homeostasis homeostasis in the in the body, body, the the disruption disruption of of which which leads leads to to various various diseases. diseases. The e essentialssential role of theserole enzymes of these in enzymes metathesis in metathesis reactions reactions in the organisms in the organisms has stimulated has stimulated interest interest in their in exploration. their exploration. Numerous reviews on nucleoside phosphorylases are available. The structural Numerous reviews on nucleoside phosphorylases are available. The structural organization of these organization of these enzymes [1,2], the substrate specificity and methods for the synthesis of enzymes [1,2], the substrate specificity and methods for the synthesis of valuable nucleosides [3–6], valuable nucleosides [3–6], and metabolism in biological systems [7–10] have been considered. and metabolismThe biosynthesis in biological of nitro systemsgenous [bases7–10] and have nucleosides been considered. in most of organisms can proceed by two Thepathways biosynthesis. De novo of synthesis nitrogenous utilizes bases some and amino nucleosides acids and insmall most organic of organisms molecules can as proceedprecursors by. two pathways.This biochemical De novo synthesispathway uses utilizes numerous some aminoenzymes acids and andit has small a high organic requirement molecules for energy. as precursors. The This biochemicalsteps of the de pathway novo synthesis uses numerous are virtually enzymes the same and in all it organisms. has a high An requirement alternative salvage for energy. pathway, The steps of thewhich de novo repeatedly synthesis utilizes are virtually fragments the of sameRNA, inis energy all organisms. saving and An italternative varies in different salvage organisms. pathway, In which repeatedlyall organisms, utilizes reactions fragments catalyzed of RNA, by is nucleoside energy saving phosphorylases and it varies play the in di keyfferent role in organisms. the salvage In all organisms,pathway reactions. The regulation catalyzed of by nucleoti nucleosidede metabolism phosphorylases and biosynthesis play the are key considered role in the in salvage relation pathway. to Escherichia coli and Salmonella enterica in [9] and in [10] for mammalian. The regulation of nucleotide metabolism and biosynthesis are considered in relation to Escherichia coli Purinergic signaling modulates fundamental pathophysiological processes, such as tissue and Salmonella enterica in [9] and in [10] for mammalian. homeostasis, wound healing, neurodegeneration, immunity, inflammation, and cancer [11]. Many Purinergiccancer processes signaling in humans modulates are accompanied fundamental by the pathophysiologicaldisturbance of pyrimidine processes, metabolism. such Colon as tissue homeostasis,carcinoma woundcells [12], healing,melanoma neurodegeneration,tumors [13], breast adenocarcinoma immunity, cells inflammation, [14], ascites hepatoma and cancer, and [11]. ManyEhrlich cancer ascites processes carcinoma in humans cells [15 are] have accompanied increased levels by theof uridine disturbance phosphorylase of pyrimidine activity. metabolism. Recent Colonexperiments carcinoma in cells mice [12 [16],] melanoma showed that tumors the disruption [13], breast of uridine adenocarcinoma homeostasis cells causes [14 cancer], ascites diseases. hepatoma, and EhrlichNucleoside ascites phosphorylase carcinoma inhibitors cells [15] provide have increased the poten levelstial to ofcorrect uridine metabolic phosphorylase defects in humans activity. [8 Recent]. experimentsThese in i micenhibitors [16] can showed also be that applied the disruption in the therapy of uridine of human homeostasis parasitic diseases. causes This cancer area diseases. of Nucleosideapplication phosphorylase is based on inhibitors the fact that provide most the of parasitespotential are to correct not able metabolic to synthesize defects purines in humans and [8]. pyrimidines de novo because their genomes lack genes encoding necessary enzymes. Hence, These inhibitors can also be applied in the therapy of human parasitic diseases. This area of significant differences between parasite and human nucleoside phosphorylases provide the basis for application is based on the fact that most of parasites are not able to synthesize purines and pyrimidines the selective inhibition of parasite enzymes. 5′-Methylthioimmucillin-H was developed as a specific de novoinhibitor because for Plasmodium their genomes falciparum lack genes PNP and encoding it was proposed necessary for enzymes. the treatment Hence, of malaria significant [17,18 di]. ffTheerences betweenhelminth parasite parasite and Schistosoma human nucleoside mansoni also phosphorylases cannot synthesize provide purines the de basis novo for [19 the]. Hence, selective the search inhibition of parasitefor inhibitors enzymes. that 5’-Methylthioimmucillin-H are specific for Schistosoma was mansoni developed PNP is as a a challenging specific inhibitor problem. for AnotherPlasmodium falciparumimportantPNP problem and it is was the proposedsearch for inhibitors for the treatmentspecific for ofGiardia malaria lamblia [17 UP,18, ].because The helminth this helminth parasite Schistosomaparasite mansoniis unablealso to synthesize cannot synthesizepyrimidines purinesde novo [ de20]. novo [19]. Hence, the search for inhibitors that are specificX-Ray crystallography for Schistosoma is mansonione of thePNP most is commonly a challenging used techniques problem. for Another studying important the structure problem- is thefunction search forrelation inhibitors of enzymes. specific In for1990,Giardia the first lamblia three-UP,dimensional because structure this helminth of human parasite erythrocytic is unable to PNP (hPNP) was determined at 3.2 Å resolution in complex with the substrate analogue 5′- synthesize pyrimidines de novo [20]. X-Ray crystallography is one of the most commonly used techniques for studying the structure-function relation of enzymes. In 1990, the first three-dimensional structure of human Biomolecules 2020, 10, 552 3 of 14 erythrocytic PNP (hPNP) was determined at 3.2 Å resolution in complex with the substrate analogue 50-iodoformycin B [21]. The enzyme molecule was found to be composed of three subunits that were related by a crystallographic threefold axis. The substrate analogue is bound in the active site at the interface between two subunits of the trimer. The substrate binding involves seven residues of one subunit and the Phe residue of the adjacent subunit. In 1995, the X-ray structure of E. coli UP was determined at 2.5 Å resolution [22]. The E. coli UP structure is a hexamer comprised three dimers that were related by a threefold axis. Each dimer is composed of two subunits related by a non-crystallographic twofold axis. The active sites of E. coli UP are formed by residues belonging to two adjacent subunits of the dimer. The structure of hexameric E. coli. PNP was established at 2.0 Å resolution in 1997 [23]. The active site of E. coli. PNP also involves residues of two adjacent subunits of the dimer. All nucleoside phosphorylases were classified into two families based on the sequence homology analysis of NPs and a comparison of their three-dimensional structures—NP-I and NP-II [1]. The NP-I family comprises hexameric and trimeric PNPs (including the MTAP subfamily) and hexameric UPs. Hexameric PNPs are enzymes from lower organisms, such as bacteria and some eukaryotic protozoa, in particular, intracellular parasites. Generally, these enzymes have a very broad spectrum of substrate specificity and are active towards both 6-amino and 6-keto derivatives of purine nucleosides [1]. Hexameric MTAPs were only isolated from the thermostable archaea Sulfolobus solfataricus [24], Pyrococcus furiosus, and Aeropyrum pernix. 5-Methylthioadenosine, adenosine, its 2-chloro and 2-amino derivatives, and also 6-keto derivatives of purine nucleosides (guanine and inosine) are substrates for the former two hexameric MTAPs [1]. 50-Deoxy-50-methylthioadenosine phosphorylase that is isolated from Aeropyrum pernix has some unique characteristics. On one hand, it is active towards some pyrimidines, including cytidine and deoxycytidine, and, on the other hand, unlike most of the reported MTAPs, it phosphorolyzes 20-fluoro-modified arabinoside [25]. Trimeric PNPs and MTAPs are found in higher organisms and bacteria. Trimeric PNPs are specific for 6-keto derivatives of purine nucleosides (guanosine and inosine, but not for xanthosine), and MTA is the main substrate for trimeric MTAPs [1]. Uridine phosphorylases from bacteria and higher organisms are generally hexamers [1]. However, Schistosoma mansoni UP is an exception [26]. This enzyme acts as a dimer. Uridine, deoxyuridine, and thymidine are substrates for UPs [1,6,27]. Meanwhile, cytidine can also serves as a substrate for S. cerevisiae UP [28]. The NP-II family includes PyNPs and TPs [1]. Thymidine phosphorylase has the highest specificity among the NPs. It is only active towards thymidine and 2’-deoxyuridine. The enzymes of the NP-II family have a dimeric quaternary structure. Monomers of nucleoside phosphorylases of the NP-II family are composed of two domains, which undergo significant rearrangements during the enzymatic reaction. High-resolution X-ray diffraction data are not available for complexes of nucleosides with nucleoside phosphorylases of the NP-II family. This review presents an analysis of conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases. Only the structures determined at 2 Å resolution or higher are considered, because atomic coordinates that are derived from lower-resolution X-ray data are not reasonably accurate to establish nucleoside conformations with certainty. The available X-ray diffraction data enable the conformational analysis of nucleosides in complexes with nucleoside phosphorylases of the NP-I family.

1.2. Structures of the Active Sites of Nucleoside Phosphorylases of the NP-I Family The structures of monomers of all nucleoside phosphorylases that belong to the NP-I family can be superimposed based on the spatially equivalent secondary structure elements. Figure1 shows the superposition of the three-dimensional structures of the monomers of hexameric PNP from B. cereus (in complex with adenosine and phosphate, PDB 3UAW, 1.20 Å [29]), trimeric PNP from Bos Taurus (in complex with immucilin H and phosphate, PDB 1B80, 1.5 Å [30]), and hexameric UP from Shewanella oneidensis (in complex with uridine and sulfate, PDB 4R2W, 1.6 Å, [31]). BiomoleculesBiomolecules2020 20,2010, ,10 552, x FOR PEER REVIEW 4 of4 15 of 14 Biomolecules 2020, 10, x FOR PEER REVIEW 4 of 15 Biomolecules 2020, 10, x FOR PEER REVIEW 4 of 15

Figure 1. Superposition of the three-dimensional structures of hexameric PNP from B. cereus (PDB Figure3UAW, 1. Superpositiongold), trimeric ofPNP the from three-dimensional Bos Taurus (PDB structures 1B80, lilac), of hexamericand hexameric PNP UP from fromB. Shewanella cereus (PDB FigureFigure 1. Superposition 1. Superposition of ofthe the three three-dimensional-dimensional structures structures ofof hexamerichexameric PNP fromfrom B.B. cereus cereus (PDB (PDB 3UAW,oneidensis gold), (PDB trimeric 4R2W PNP, green from). TheBos structures Taurus (PDBare superimposed 1B80, lilac), with and the hexameric COOT program. UP from Shewanella 3UAW,3UAW, gold gold), trimeric), trimeric PNP PNP from from Bos Bos Taurus Taurus (PDB (PDB 1B80, 1B80, lilac),lilac), andand hexameric UPUP fromfrom Shewanella Shewanella oneidensis (PDB 4R2W, green). The structures are superimposed with the COOT program. oneidensisToneidensishe active (PDB (PDB sites 4R2W 4R2W of ,these green, green three). The). The enzymes structures structures are are are well superimposed superimposed superimposed withwith, a thethes can COOT be seen program.program. in Fig ure 1. Figures 2–The4 show active the active sites ofsites these of these three enzymes enzymes (the are orientation well superimposed, is the same as in as Fig canure be 1). seen in Figure1. TheT activehe active sites sites of theseof these three three enzymes enzymes are are well well superimposed superimposed,, as can be seenseen in in Fig Figureure 1 1. Figures. Figures Figures2–4 show the active sites of these enzymes (the orientation is the same as in Figure1). 2–4 2show–4 show the the active active sites sites of ofthese these enzymes enzymes (the (the orientation orientation is is thethe samesame as in FigFigureure 1). 1).

Figure 2. Structure of the active site of hexameric B. cereus PNP in complex with adenosine and sulfate Figure 2. Structure of the active site of hexameric B. cereus PNP in complex with adenosine and sulfate (PDB 3UAW). The nitrogen, oxygen, and sulfur atoms are shown in blue, red, and yellow, Figure 2. Structure of the active site of hexameric B. cereus PNP in complex with adenosine and sulfate (PDBrespectively. 3UAW). The The nitrogen, carbon atoms oxygen, of the and protein sulfur and atoms the aresubstrate shown are in gold blue, and red, violet, and yellow, respectively. respectively. The Figure(PDB 2. Structure 3UAW). Theof the nitrogen, active site oxygen, of hexameric and sulfur B. cereus atoms PNP are in complex shown inwith blue, adenosine red, and and yellow, sulfate Theatoms carbon of the atoms adjacent of the subunit protein of and thethe PNP substrate dimer are are colored gold and in grey. violet, The respectively. hydrogen bonds The atoms involving of the (PDBrespectively. 3UAW). The carbon nitrogen, atoms oxygen, of the protein and sulfur and the atoms substrate are are shown gold and in blue,violet, red,respectively. and yellow, The adjacentthe adenosine subunit molecule of the PNP and dimer sulfate are are colored indicated in grey.by das Thehed hydrogenlines. bonds involving the adenosine respectively.moleculeatoms andof the The sulfate adjacent carbon are subunitatoms indicated of of the bythe protein dashedPNP dimer and lines. arethe coloredsubstrate in aregrey. gold The and hydrogen violet, bondsrespectively. involving The atomsthe ofadenosine the adjacent molecule subunit and ofsulfate the PNP are indicated dimer are by colored dashed inlines. grey. The hydrogen bonds involving the adenosine molecule and sulfate are indicated by dashed lines.

Figure 3. Structure of the active site of Shewanella oneidensis MR-2 UP in complex with uridine and sulfateFigure (PDB 3. Structure 4R2W). of The the nitrogen, active site oxygen, of Shewanella and sulfur oneidensis atoms MR are-2 UP shown in complex in blue, with red, uridine and yellow, and respectively.sulfate (PDB The 4R2W). carbon The atoms nitrogen, of the oxygen, protein andand thesulfur substrate atoms are are greenshown and in blue, violet, red, respectively. and yellow, The Figure 3. Structure of the active site of Shewanella oneidensis MR-2 UP in complex with uridine and atomsrespectively. of the adjacent The carbon subunit atoms of theof the UP protein dimer an ared the colored substrate in grey. are green The hydrogen and violet, bonds respectively. involving The the sulfate (PDB 4R2W). The nitrogen, oxygen, and sulfur atoms are shown in blue, red, and yellow, atoms of the adjacent subunit of the UP dimer are colored in grey. The hydrogen bonds involving the Figureuridinerespectively. 3 molecule. Structure The and ofcarbon sulfatethe active atoms are indicatedsiteof the of proteinShewanella by dashedand theoneidensis lines.substrate MR are-2 greenUP in and complex violet, respectively.with uridine The and uridine molecule and sulfate are indicated by dashed lines. sulfateatoms (PDB of the 4R2W). adjacent The subunit nitrogen, of the oxygen, UP dimer and are sulfur colored atoms in grey. are The shown hydrogen in blue, bonds red, involving and yellow, the respectively.uridine molecule The carbon and sulfateatoms ofare the indicated protein by an dashedd the substrate lines. are green and violet, respectively. The atoms of the adjacent subunit of the UP dimer are colored in grey. The hydrogen bonds involving the uridine molecule and sulfate are indicated by dashed lines. Biomolecules 2020, 10, x FOR PEER REVIEW 5 of 15 Biomolecules 2020, 10, 552 5 of 14

Figure 4. Structure of the active site of trimeric Bos Taurus PNP (PDB 1B80). The nitrogen, oxygen, and Figuresulfur 4. atomsStructure are shownof the inactive blue, site red, of and trimeric yellow, Bos respectively. Taurus PNP The (PDB carbon 1B80). atoms The of nitrogen, the protein oxygen, and the andsubstrate sulfur atoms are lilac are and shown violet, in respectively.blue, red, and The yellow, hydrogen respectively. bonds involving The carbon the atoms immucillin of the H protein molecule andand the phosphate substrate are indicatedlilac and violet, by dashed respectively. lines. The hydrogen bonds involving the immucillin H molecule and phosphate are indicated by dashed lines. In all enzymes, the base of the nucleoside is bound in the hydrophobic pocket and it forms hydrogenIn all enzymes, bonds with the the base residues of the nucleoside of the enzyme. is bound In all in cases, the hydrophobic adjacent subunits pocket of and the oligomericit forms hydrogenmolecules bonds are involved with the in residues the complex of the formation enzyme. with In all nucleosides. cases, adjacent In the subunits ﬁgures, theof adjacentthe oligomeric subunits moleculesof the hexameric are involved PNP and in the UP complex are colored formation in grey. withFor the nucleosides. trimeric PNP, In the adjacent figures, subunitthe adjacent is only subunitsinvolved of inthe the hexameric formation PNP of the and hydrophobic UP are colored pocket in grey and. itFor is notthe trimeric shown in PNP, Figure the4. adjacent The ribose subunit moiety is andonly phosphate involved in (or the sulfate) formation are bound of the in hydropho a similar fashionbic pocket in the and hexameric it is not PNPshown and in UP. Figure This 4. is dueThe to ribosethe similar moiety structures and phosphate of the phosphate-(or sulfate) orare sulfate-binding bound in a similar subsites fashion of these in the enzymes hexameric and the PNP presence and UP.of aThis conserved is due glutamate to the similar residue structures that forms of hydrogen the phosphate bonds- withor sulfate the O2-0bindingand O3 0 subsiteatomss of of the these ribose enzymesmoiety. and The th radicallye presence diﬀ oferent a conserved binding ofglutamate the ribose residue moiety that and forms phosphate hydrogen (or sulfate) bonds with is observed the O2′ in andthe O trimeric3′ atoms PNP. of the ribose moiety. The radically different binding of the ribose moiety and phosphate (or sulfate) is observed in the trimeric PNP. 1.3. Description of Nucleoside Conformations

1.3. Description of Nucleoside Conformationsi The parameters P, θm, χ, ν2, and Tj that are given in Tables 1–3 adequately and fully characterize nucleosideThe parameters conformations. P, θm, χ, ν2 The, and conformation iTj that are given of thein Tables pentofuranose 1–3 adequately ring can and be fully described characterize by the nucleosidefollowing conformations. two parameters: The the conformation phase angle of of pseudorotation the pentofuranoseP and ring the amplitude can be described of pseudorotation by the followingθm. These two parameters parameters: are the calculated phase angle from of pseudorotation the endocyclic torsionP and the angles amplitudeνi by theof pseudorotation equations given θmin. These [32]. Theparameters tables also are includecalculated the from endocyclic the endocyclic torsion angletorsionν 2angles(C10–C2 νi 0by–C3 the0–C4 equations0), which given provides in [32information]. The tables on also the degree includ ofe the staggering endocyclic of substituents torsion an atgle the ν2 C2 (C0 1′and–C2′ C3–C0 atoms3′–C4′), of which the pentofuranose provides informationring. The on mutual the degree orientation of staggering of the pentofuranose of substituents ring at the and C2′ the and heterocyclic C3′ atoms of base the ispentofuranose determined by ring.the T glycosidiche mutual angle orientationχ (C4pur of (C2the pyrpentofuranose)–N9pur (N1 pyrring)–C1 and0–O4 the0 ),heterocyclic according tobase the is IUPAC–IUB determined rules by the [33 ]. glycosidicTwo regions angle of χ the(C4pur angle (C2pyrχ )are–N9 distinguished:pur (N1pyr)–C1′–Osyn4′),(0 according90 ) andto theanti IUPAC(180 –IUB90 rules). Additionally, [33]. Two ◦ ± ◦ ◦ ± ◦ regionshighlight of the areas angle high χ synare (distinguished:χ from 90 until syn ~120 (0° ±in 90anti°) andregion) anti and(180° high ± 90anti°). Additionally(χ from 90 , highlightuntil ~ 60 ◦ ◦ − ◦ − ◦ areasin syn highregion). syn (χ Scheme from 902 °shows until ~120 these° fourin anti regions region) of andχ for high uridine. anti (χ from −90° until ~−60° in syn region).The Scheme phase 2 shows angle these of pseudorotation four regions of can χ for be uridine related. to the description of the conformation of the furanose ring that is based on displacements of atoms from the mean plane of the ring. The pentofuranose ring is generally non-planar and it can adopt an envelope conformation (iE), i in which four atoms lie in a single plane, or a twist conformation ( Tj), in which two adjacent atoms are displaced in the opposite directions from the mean plane of the ring. The indices correspond to the numbers of the atoms that are displaced from the mean plane of the ring, with the index 0 referring to the O4’ atom. The indices of the atoms that are displaced towards the C4’–C5’ bond are given as superscripts. The twist conformations are denoted by placing the index of the atom most displaced from the mean plane of the ring before the letter T, while the adjacent atom less displaced from the mean plane follows T. Biomolecules 2020, 10, 552 6 of 14 Biomolecules 2020, 10, x FOR PEER REVIEW 6 of 15

Biomolecules 2020, 10, x FOR PEER REVIEW 9 of 15 Scheme 2. The mutual orientation of the pentofuranose ring and the heterocyclic base for uridine are SchemeIn 43 2. complexes,The mutual purine orientation bases of adopt the pentofuranose a high-syn (χ is ring in the and range the heterocyclic of 93–120°) orientationbase for uridine or they are anti syn anti syn shown:shown:are in the aandnti immediate and syn(above, (above, vicinity side side to view) viewthis conformational) and and highhigh anti andregion high high (χ syn is in (below, (below,the range view view of from 127 from– above131 above).°). ).In all of these structures, two pairs of atoms of the nucleosides (N3 and C2′; C8 and O4′) form rather short TheThe anglecontacts phase of (about pseudorotationangle 3 Å of ). In pseudorotation the other of four each complexes can unsymmetrical be related (these are to four thetwist ofdescription the seven conformation complexes of the conformation with varies formycin over of the a range of 18furanose◦ (FigureA in the ring5). structure Therefore,that is 6F based4X; theTable on character 1, displacements complexes of 1, atomic2, 4, of and atoms 6), displacements nucleosides from the adopt mean in a eachhigh plane-anti of of orientation 20 the sectors ring. The of the pseudorotationpentofuranose(χ varies cycle in ring the is range is preserved, generally from −67 non i.e.,° to -−planar the71°). numbersIn andthese it conformers, can of theadopt atoms there an envelope are displaced short contactsconformation from between the mean (i Ethe), inN8 plane which of the and C2′ atoms (the interatomic distance is about 3 Å ). No correlationi between the angle χ and ring,four the directions atoms lie in of athese single displacements, plane, or a twist and conformation the ratio of the ( Tj) displacements, in which two (inadjacent magnitude) atoms are remain displacedpresence in the of a opposite sulfate or directionsphosphate ionfrom in thethe complex mean plane was revealed. of the ring. The indices correspond to the unchanged. EitherPseudorotation symmetrical angles twist of purine conformations nucleosides and or envelope-typetheir analogs in the conformations active sites of hexameric bound the sectors numbers of the atoms that are displaced from the mean plane of the ring, with the index 0 referring of the pseudorotationPNPs are shown cycle. in Figure 5 in red in the presence of sulfate (phosphate) ions, and in green when to the O4′ atom. The indices of the atoms that are displaced towards the C4′–C5′ bond are given as sulfate (phosphate) ions are absent. superscripts. The twist conformations are denoted by placing the index of the atom most displaced from the mean plane of the ring before the letter T, while the adjacent atom less displaced from the mean plane follows T. The angle of pseudorotation of each unsymmetrical twist conformation varies over a range of 18° (Figure 5). Therefore, the character of atomic displacements in each of 20 sectors of the pseudorotation cycle is preserved, i.e., the numbers of the atoms displaced from the mean plane of the ring, the directions of these displacements, and the ratio of the displacements (in magnitude) remain unchanged. Either symmetrical twist conformations or envelope-type conformations bound the sectors of the pseudorotation cycle.

1.4. Conformations of Free Nucleosides The conformations of nucleosides in the free form were studied in detail. The frequency of the occurrence of the parameters P and θm in crystals of 178 nucleosides was analyzed in [34]. The phase angles P of nucleosides were found in two ranges, from −1° to 34° and from 137° to 194°. The former range belongs to the N region (P varies from −45° to 45°), and the latter one to the S region (P is in the range of 135–225°). Only five nucleoside derivatives were found in the range from 84° to 106°, belonging to the E region (P is in the range of 45–135°). No conformers were found in the W region (P varies from 225° to −45°). The amplitude of pseudorotation θm of nucleosides in the crystal structures have a unimodal distribution over the range from 30° to 46°. It is almost independent of the type of the base and the phase angle P. The more planar the furanose ring, the smaller the parameter θm. i i FigureThe 5. resultsFigurePseudorotation 5. ofPseudorotation which cyclewere cycle ofanalyzed of the the pentofuranose pentofuranose in [35], the ring N ring in and nucleosides. in S nucleosides. conformers Sectors are Sectorsof named nucleosides as are Tj and named exist as in Taj j iTj for unsymmetrical twist conformations (the character of atomic displacements from the mean plane anddynamiciT for equilibrium unsymmetrical, according twist conformations to extensive NMR (the characterexperiments of atomic in solution displacements. The percentage from the ratio mean of plane of theof the ring ring is is preserved preserved in ineach each sector). sector). Pseudorotation Pseudorotation angles of purine angles nucleosides of purine and nucleosides their analogs and their the N andin S theconformers active sites existingof hexameric in equilibriumPNPs in the presence in solution of sulfate depend (phosphate)s on theions nature are shown of thein red, nucleic acid analogsbase (purine inwhile the oractivewhen pyrimidine) sulfate sites of(phosphate) hexameric and theions PNPs typeare absent inof the thethey presence furanoseare shown of in ring sulfate green. (ribose Pseudorotation (phosphate) or deoxyribose). angles ions are of shown Purine in red,nucleosides whilepurine when prefer nucleosides sulfate the S (phosphate) conformerand their analogs, ionswhile in are thepyrimidine absentactive sites they ofnucleosides aretrimeric shown PNPs prefer in in green. the thepresencePseudorotation N conformation. of sulfate angles The of purine nucleosides(phosphate) ions and are theirshown analogsin violet, while in the when active sulfate sites (phosphate) of trimeric ions PNPsare absent in they the presenceare shown of sulfate (phosphate)in blue. ions Pseudoratation are shown in angles violet, of while uridine when or thymidine sulfate in (phosphate) the active sites ions of UP are in absent the presence they are of shown in sulfate ions are shown as yellow, while when sulfate ions are absent they are shown as light pink. blue. Pseudoratation angles of uridine or thymidine in the active sites of UP in the presence of sulfate

ions are shownThe puckering as yellow, amplitudes while when θm of sulfateall conformers ions are in absentthe active they sites are of shownhexameric as lightPNPs pink.and MTAP (except for the structure 4D9H) are in the range of 17–35°. In the structure 4D9H, the nucleoside adopts a conformation with a nearly planar furanose ring (θm = 4°), which is not typical of nucleosides. This might be attributed to the fact that the nucleoside with one-half occupancy is only present in one of the two crystallographically independent molecules. It is worth noting that not all atoms of the nucleoside are well fitted to the 2F0–Fc electron density at the 1σ level. Hence, the conclusions of the study [47] should be considered with caution. Biomolecules 2020, 10, 552 7 of 14

1.4. Conformations of Free Nucleosides The conformations of nucleosides in the free form were studied in detail. The frequency of the occurrence of the parameters P and θm in crystals of 178 nucleosides was analyzed in [34]. The phase angles P of nucleosides were found in two ranges, from 1 to 34 and from 137 to 194 . The former − ◦ ◦ ◦ ◦ range belongs to the N region (P varies from 45 to 45 ), and the latter one to the S region (P is in − ◦ ◦ the range of 135–225◦). Only ﬁve nucleoside derivatives were found in the range from 84◦ to 106◦, belonging to the E region (P is in the range of 45–135◦). No conformers were found in the W region (P varies from 225 to 45 ). The amplitude of pseudorotation θm of nucleosides in the crystal structures ◦ − ◦ have a unimodal distribution over the range from 30◦ to 46◦. It is almost independent of the type of the base and the phase angle P. The more planar the furanose ring, the smaller the parameter θm. The results of which were analyzed in [35], the N and S conformers of nucleosides exist in a dynamic equilibrium, according to extensive NMR experiments in solution. The percentage ratio of the N and S conformers existing in equilibrium in solution depends on the nature of the nucleic acid base (purine or pyrimidine) and the type of the furanose ring (ribose or deoxyribose). Purine nucleosides prefer the S conformer, while pyrimidine nucleosides prefer the N conformation. The N S ↔ conformational interconversions occur through the E region of the pseudorotation cycle (at about P = 90 ), because the energy barrier in this region is lower. The E barrier for the N S interconversion ◦ ↔ occurs due to steric strain that was caused by the eclipsed orientation of substituents at the C20 and C30 atoms at P = 90◦. For the alternative pathway through the W region in the vicinity of the phase angle P = 270◦, the energy barrier is much higher. In this region, in addition to the steric strain between the substituents at the C20 and C30 atoms, there is also steric strain between the nitrogenous base and the exocyclic 50-oxymethyl group due to their close arrangement of these bulky substituents. Diﬀerent computational methods evaluated the energies of barriers to pseudorotation of derivatives of the furanose ring in the W and E regions. The analysis of the results of these calculations provided the estimate of their average values [36]. The barrier of pseudorotation in the W region is ~24 kJ/mol for 20-deoxyribofuranose and ~31 kJ/mol for ribofuranose, according to these estimations. The E region is characterized by the barrier of ~7.5 kJ/mol for 20-deoxyribofuranose and ~16 kJ/mol for ribofuranose. The barrier of pseudorotation of purine nucleosides in the E region, which was experimentally evaluated from the 13C NMR data, is 20 2 kJ/mol [37]. It was suggested that the energy barrier for pyrimidine ± nucleosides is higher than 25 kJ/mol. In the solutions, purine nucleosides occur with equal frequency in syn- and anti-conformations, whereas pyrimidine nucleosides more often exist in the anti-conformation [35,36]. The rotation about the glycosidic bond is not free [38]. In the case of pyrimidine nucleosides, the interconversions between these conformations are associated with steric strain between the protons at the C2’ and C6 atoms in the χ angle range near the high-anti region (from 60 to 90 ) and steric interaction − ◦ − ◦ between the proton at the C2’ atom and O2 atom in the χ angle range in the vicinity of the high-syn region (90–120 ). The energy of the barrier to syn anti transitions near the high-syn region was ◦ ↔ experimentally evaluated at 43–46 kJ/mol from the analysis of NMR spectroscopic data for pyrimidine nucleosides [39].

2. Discussion

2.1. Conformations of Nucleosides and Their Analogues in the Active Sites of PNPs and MTAP with Hexameric Quaternary Structure Table 1 presents the conformational parameters of nucleosides in 47 complexes, including complexes with hexameric PNPs from B. cereus, Vibrio cholera, Helicobacter pylori, E. coli, Toxoplasma Biomolecules 2020gondii, 10,, 552and B. subtillis, and complexes with hexameric MTAP from Sulfolobus solfataricus. In 37 8 of 14 complexes, the active sites contain a sulfate (phosphate) ion, and in 10 complexes this ion is absent. It should be noted that there are unacceptably short interatomic contacts and non-standard bond nucleosidesangles in the of structure nucleosides of inHelicobacter the structure pyloriof HelicobacterPNP in pylori complex PNP in withcomplex formycin with formy A (PDBcin A (PDB 6F4X). Hence, we re-reﬁned6F4X). this Hence, structure we re to-refined calculate this the structure conformational to calculate theparameters conformational of the parameters nucleoside. of the nucleoside.

Table 1. ConformationsTable 1. Conformations of nucleosides of nucleosides and and their their analogues analogues in in the the active active sites sitesof hexameric of hexameric PNPs and PNPs and 50-deoxy-505′-methylthioadenosine-deoxy-5′-methylthioadenosine phosphorylases phosphorylases (MTAP).(MTAP).

PDB Res. θm Organism Enzyme Complex PDB Ref.. Shape P deg. θνm2 deg. νχ2 deg. Organism Enzyme Complex ID Res. Ǻ Ref. Shape P deg.deg. χ deg. ID deg. deg. B. cereus AdoP; w.t. Ado, SO4 3UAW 1.20 [29] 4T3 229 30 −20 +103 4 B. cereus B. cereusAdoP; w.t.AdoP; w.t. Ado, SO4Ino, SO4 3UAW3UAX 1.201.20 [ 29]] 4T3 T 226 22930 30−21 20+101 +103 3 − B. cereus AdoP; w.t.AdoP; Ino, SO4 3UAX 1.20 [29] 4T 226 30 21 +101 3 − B.AdoP; cereus D204ND204N Ado, SO4 3UAY 1.40 [29] 4T3 229 28 −18 +102 B. cereus Ado, SO4 3UAY 1.40 [29] 4T 229 28 18 +102 mutant mutant 3 − AdoP; D204NAdoP; 4 B. cereus Ino, SO4 3UAZ 1.40 [29] T3 223 29 21 +99 B. cereusmutant D204N Ino, SO4 3UAZ 1.40 [29] 4T3 223 29 −21 − +99 1 mutant OT 275 27 +2 +94 1 1 OT O T 275 27727 28+2 +3+94 +93 OT1 T1 277 26728 29+3 1+93 +95 V. cholera V. choleraPNP; w.t.PNP; w.t. Ado Ado 1VHW1VHW 1.541.54 [40]] O OT1 1 267 29 −1 −+95 OT 276 26 +3 +93 OT1 1 276 26 +3 +93 OT 269 27 0 +95 1 OT 273 30 +2 +97 1T / T4 305/258 17/29 9.9/ 6.2 67/+118 O O − − formycin A 1T 308 33 +20 69 2 − PO4 (in T4 254 33 9 +127 H. pylori PNP; w.t. 6F4X 1.694 [41] O − monomers 3, 5 1T 318 30 +22 71 2 − and 6) 4T 230 32 20 +120 3 − T1 278 28 +4 67 O − 4T 226 30 21 +105 B. cereus AdoP; w.t. Ado, SO4 2AC7 1.7 [42] 3 − 4T 227 29 20 +106 3 − T4 212 24 20 +107 formycin A, 3 − E. coli PNP; w.t. 4TS9 1.77 [43] T4 213 25 21 +108 PO4 3 − 4T 220 28 21 +116 3 − 4T 224 23 16 +107 3 − 4T 222 25 19 +103 3 − S. 4T 221 23 17 +107 MTAP; w.t. Guo, SO4 1JE1 1.8 [24] 3 − solfataricus 4T 225 28 20 +109 3 − 4T 226 27 18 +106 3 − 4T 229 28 18 +108 3 − PNP D204A, 4T 236 28 16 +106 formycin A, O − E. coli R217A double 4TTJ 1.87 [43] 4T 223 30 22 +115 PO4, SO4 3 − mutant 4T 241 27 13 +105 O − T4 206 28 25 +104 formycin A, 3 − E. coli PNP; w.t. 3UT6 1.89 [44] 4T 220 26 20 +106 PO4 3 − 4T 221 30 22 +114 3 − 4T 241 34 17 +116 2-ﬂuoro Ado, O − E. coli PNP; w.t. 1PK9 1.9 [45] 4T 236 35 19 +106 PO4 O − 4T 236 32 18 +106 O − immucillin H, 4T 234 32 19 +130 T. gondii PNP; w.t. 3MB8 1.9 [46] O − PO4 4T 232 34 21 +130 3 − BsPNP-233; B. subtilis Ado 4D9H 1.91 [47] 3T 4 4 +4 +107 w.t. 2 T4 267 32 2 +131 S. O − MTAP; w.t. MTA, SO4 1JDT 2.0 [24] T4 254 30 9 +127 solfataricus O − T4 259 28 5 +120 O − 4T 237 28 15 +115 O − S. 4T 238 27 14 +119 MTAP; w.t. Ado, SO4 1JDV 2.0 [24] O − solfataricus 4T 238 28 15 +120 O − 4T 237 28 15 +114 O − Note: there are two variant structures of complexes of the ﬁrst monomer of structure 6F4X, which are separated by “/”.

Table1 shows that the phase angles P of all the nucleosides vary over a wide part of the left part of the pseudorotation cycle (206–318◦). The following two ranges of P values can be distinguished: (206–254◦) and (258–318◦). In the structures of the complexes containing a sulfate or phosphate ion, the nucleosides adopt a conformation that corresponds to the former range. The exceptions are two complexes of MTA with MTAP that are characterized by slightly larger angles P (259◦ and 267◦). 4 4 4 The conformations in the former region belong to one of the following four types: 3T , T3, TO, and 4 4 OT . Only the 3T conformation is observed in the crystals of free nucleosides [34]. In the complexes, in which a sulfate (phosphate) ion is absent in the active site, namely, six complexes in the structure Biomolecules 2020, 10, 552 9 of 14

1VHW, three complexes in the structure 6F4X (monomers 1, 2, and 4), and one of the complexes in 6F4X, in whichBiomolecules phosphate 2020, 10, x FOR is present PEER REVIEW with one-half occupancy (monomer 6), the angle P7is of in15 the range of 258–318 . These are the T4, T1, 1T , and 1T conformers. These conformers were not found ◦NS conformationalO interconversionsO O occur through2 the E region of the pseudorotation cycle (at 4 1 1 for nucleosidesabout inP = the 90° free), because form. the In energy three barrier of them in this (OT region, OT is, and lower. TTheO), E the barrier endocyclic for the N oxygenS atom is displacedinterconversion from the mean occurs plane due to steric of the strain ring that in was the caused direction by the eclipsed opposite orientation to the of glycosidic substituents bond and at the C2′ and C3′ atoms at P = 90°. For the alternative pathway through the W region in the vicinity4 1 the C40–C50 bond. This displacement of the O40 atom (that is most pronounced in the OT and OT of the phase angle P = 270°, the energy barrier is much higher. In this region, in addition to the steric conformers)strain results between in these the substituents bonds having at the axial C2′ and positions. C3′ atoms, As there mentioned is also steric above, strain the between potential the energy of these nucleosidenitrogenous conformations base and the exocyclic is high 5′ due-oxymethyl to steric group strain due atto theirP = close270◦ arrangement(W region). of these bulky In 43 complexes,substituents. purine bases adopt a high-syn (χ is in the range of 93–120◦) orientation or they Different computational methods evaluated the energies of barriers to pseudorotation of are in the immediate vicinity to this conformational region (χ is in the range of 127–131◦). In all of derivatives of the furanose ring in the W and E regions. The analysis of the results of these calculations these structures,provided two the pairsestimate of of atoms their average of the values nucleosides [36]. The barrier (N3 and of pseudorotation C2’; C8 and in O4’)the W form region rather is short contacts (about~24 kJ/mol 3 Å). for In 2′ the-deoxyribofuranose other four complexes and ~31 kJ/mol (these for areribofuranose four of,the according seven to complexes these estimations with. formycin A in the structureThe E region 6F4X; is characterized Table1, complexes by the barrier 1, 2, of 4, ~7.5 and kJ/mol 6), nucleosides for 2′-deoxyribofuranose adopt a high- and ~16anti kJ/molorientation (χ for ribofuranose. The barrier of pseudorotation of purine nucleosides in the E region, which was varies in the range from 67◦ to 71◦). In these conformers, there are short contacts between the N8 experimentally evaluated− from− the 13C NMR data, is 20 ± 2 kJ/mol [37]. It was suggested that the and C2’ atomsenergy (the barrier interatomic for pyrimidine distance nucleosides is about is higher 3 Å). than No 25 correlation kJ/mol. between the angle χ and presence of a sulfate or phosphateIn the solutions, ion purine in the nucleosides complex occur was with revealed. equal frequency in syn- and anti-conformations, Pseudorotationwhereas pyrimidine angles nucleosides of purine more nucleosides often exist in and the theiranti-conformation analogs in [35,36 the]. activeThe rotation sites about of hexameric the glycosidic bond is not free [38]. In the case of pyrimidine nucleosides, the interconversions PNPs are shown in Figure5 in red in the presence of sulfate (phosphate) ions, and in green when between these conformations are associated with steric strain between the protons at the C2’ and C6 sulfate (phosphate)atoms in the ions χ angle are range absent. near the high-anti region (from −60° to −90°) and steric interaction between The puckeringthe proton amplitudesat the C2’ atomθ andm ofO2 allatom conformers in the χ angle in range the in active the vicinity sites of of the hexameric high-syn region PNPs (90 and– MTAP 120°). The energy of the barrier to syn  anti transitions near the high-syn region was experimentally (except for the structure 4D9H) are in the range of 17–35◦. In the structure 4D9H, the nucleoside evaluated at 43–46 kJ/mol from the analysis of NMR spectroscopic data for pyrimidine nucleosides adopts a conformation[39]. with a nearly planar furanose ring (θm = 4◦), which is not typical of nucleosides. This might be attributed to the fact that the nucleoside with one-half occupancy is only present in one of the two crystallographically2. Discussion independent molecules. It is worth noting that not all atoms of the σ nucleoside2.1. are C wellonformations ﬁtted of to N theucleosides 2F0–Fc and electronTheir Analogues density in the at Active the S 1iteslevel. of PNPs Hence, and MTAP the with conclusions of the study [47] shouldHexameric be Quaternary considered Structure with caution. Table 1 presents the conformational parameters of nucleosides in 47 complexes, including 2.2. Conformationscomplexes of Nucleosideswith hexameric in PNPs the Active from B. Sites cereus of, Vibrio PNPs cholera and MTAP, Helicobacter with pylori Trimeric, E. coli Quaternary, Toxoplasma Structure Table2gondii presents, and B. the subtillis conformational, and complexes parameters with hexameric of MTAP nucleosides from Sulfolobus in complexes solfataricus. with In 37 trimeric complexes, the active sites contain a sulfate (phosphate) ion, and in 10 complexes this ion is absent. MycobacteriumIt should tuberculosis be noted, Schistosomathat there are mansoniunacceptably, and short calf interatomic spleen PNPs, contacts and and in non the-standard complex bond with trimeric MTAP fromangles human of nucleosides placenta. in Therethe structure are aof total Helicobacter of nine pylori complexes, PNP in complex eight with of whichformycin contain A (PDB a sulfate (phosphate)6F4X). ion. Hence, we re-refined this structure to calculate the conformational parameters of the nucleoside.

Table 2. ConformationsTable 1. Conformations of nucleosides of nucleosides andand their their analogues analogues in the active in the sites active of hexameric sites PNPs of trimeric and PNPs and MTAP. 5′-deoxy-5′-methylthioadenosine phosphorylases (MTAP).

PDB Res. θm Organism Enzyme Complex PDB Ref.. Shape P deg. θνm2 deg. νχ2 deg. Organism Enzyme Complex ID Res. Ǻ Ref. Shape P deg.deg. χ deg. ID deg. deg. B. cereus AdoP; w.t. Ado, SO4 3UAW 1.20 [29] 4T3 229 30 −20 +103 B. cereus AdoP; w.t.Immucillin Ino, H, SO4 3UAX 1.20 [29] 4T3 4 226 30 −21 +101 Bos taurus PNP; w.t. 1B80 1.5 [30] TO 239 29 15 +133 AdoP; PO4 − 4 H sapiens B. cereusMTAP; w.t.D204N MTA, SO4Ado, SO4 1CG63UAY 1.71.40 [ 2948]] 4T3 TO 229 24128 32−18 15+102 +126 4 − mutant 3T 215 20 16 +129 M. Immucillin H, 4 − PNP; w.t. AdoP; 1G2O 1.75 [49] T3 217 18 15 +130 tuberculesis PO4 4 4 − B. cereus D204N Ino, SO4 3UAZ 1.40 [29] T3 3T 223 21429 21−21 17+99 +128 mutant 4 − Ino, and SO4 TO 238 28 15 +131 OT1 2 275 27 +2 − +94 S. mansony PNP; w.t. in monomers 3FAZ 1.9 [50] 1T 136 12 8 +113 OT14 277 28 +3 −+93 V. cholera PNP; w.t. 1 and 3Ado 1VHW 1.54 [40] TO 237 28 16 +131 OT14 267 29 −1 − +95 Calf splean PNP; w.t. Ino, SO4 1A9S 2.0 [51] TO 243 27 12 +133 OT1 276 26 +3 − +93

The phase angles P of all the nucleosides in the complexes containing a sulfate (phosphate) ion are in the range of 214–243◦, which is narrower than the range of P for nucleosides in complexes with hexameric PNPs, as it is seen from the Table; the angle χ is in the range of 126–133◦ and corresponds to the anti region in the immediate vicinity of the high-syn region. These structures also have rather Biomolecules 2020, 10, x FOR PEER REVIEW 7 of 15

NS conformational interconversions occur through the E region of the pseudorotation cycle (at about P = 90°), because the energy barrier in this region is lower. The E barrier for the NS interconversion occurs due to steric strain that was caused by the eclipsed orientation of substituents at the C2′ and C3′ atoms at P = 90°. For the alternative pathway through the W region in the vicinity of the phase angle P = 270°, the energy barrier is much higher. In this region, in addition to the steric strain between the substituents at the C2′ and C3′ atoms, there is also steric strain between the nitrogenous base and the exocyclic 5′-oxymethyl group due to their close arrangement of these bulky substituents. Different computational methods evaluated the energies of barriers to pseudorotation of derivatives of the furanose ring in the W and E regions. The analysis of the results of these calculations provided the estimate of their average values [36]. The barrier of pseudorotation in the W region is ~24 kJ/mol for 2′-deoxyribofuranose and ~31 kJ/mol for ribofuranose, according to these estimations. The E region is characterized by the barrier of ~7.5 kJ/mol for 2′-deoxyribofuranose and ~16 kJ/mol for ribofuranose. The barrier of pseudorotation of purine nucleosides in the E region, which was experimentally evaluated from the 13C NMR data, is 20 ± 2 kJ/mol [37]. It was suggested that the energy barrier for pyrimidine nucleosides is higher than 25 kJ/mol. In the solutions, purine nucleosides occur with equal frequency in syn- and anti-conformations, Biomolecules 2020, 10, 552 10 of 14 whereas pyrimidine nucleosides more often exist in the anti-conformation [35,36]. The rotation about the glycosidic bond is not free [38]. In the case of pyrimidine nucleosides, the interconversions between these conformations are associated with steric strain between the protons at the C2’ and C6 short interatomicatoms contacts in the χ angle similar range tonear those the high found-anti region in hexameric (from −60° to − PNPs.90°) and Thesteric puckeringinteraction between amplitudes θm the proton at the C2’ atom and O2 atom in the χ angle range in the vicinity of the high-syn region (90– are about 25◦. 120°). The energy of the barrier to syn  anti transitions near the high-syn region was experimentally In one ofevaluated the three at 43 complexes–46 kJ/mol from of inosinethe analysis with of NMRShistosoma spectroscopic mansony data for pyrimidinePNP (PDB nucleosides 3FAZ), a sulftate ion is absent.[39 In]. this complex, inosine adopts another conformation: χ is in the high-syn region (χ = 113◦), the furanose ring is much more planar, and the phase angle belongs to the right part of the 2. Discussion pseudorotation cycle (P = 136◦). Pseudorotation2.1. Conformations angles of of N purineucleosides nucleosides and Their Analogues and in their the A analogsctive Sites of in PNPs the and active MTAP sites with of trimeric PNPs in Figure5 are shownHexameric in Q violetuaternary in Structure the presence of sulfate (phosphate) ions and, when sulfate (phosphate) ions are absent, theyTable are 1 shownpresents the in blue.conformational parameters of nucleosides in 47 complexes, including complexes with hexameric PNPs from B. cereus, Vibrio cholera, Helicobacter pylori, E. coli, Toxoplasma gondii, and B. subtillis, and complexes with hexameric MTAP from Sulfolobus solfataricus. In 37 2.3. Conformationscomplexes, of Nucleosides the active sites in thecontain Active a sulfate Sites (phosphate) of UPs ion, and in 10 complexes this ion is absent. It should be noted that there are unacceptably short interatomic contacts and non-standard bond Table3 presentsangles of the nucleosides conformational in the structure parameters of Helicobacter of pylori nucleosides PNP in complex in 14 with complexes formycin A with (PDB UPs from Vibrio cholera, Shewanella6F4X). Hence, oneidensis we re-refined, Schistosoma this structure mansoni to calculate, and theSalmonella conformational typhimurium parameters . of Six the of these 14 complexes do notnucleoside contain. sulfate ions.

Table 1. Conformations of nucleosides and their analogues in the active sites of hexameric PNPs and 5′-deoxyTable-5′ 3.-methylthioadenosineThe conformations phosphorylases of nucleosides (MTAP). in the active sites of UPs.

PDB Res. θm Organism Enzyme Complex PDB Ref.. Shape P deg. θνm2 deg. νχ 2deg. Organism Enzyme Complex ID Res. Ǻ Ref. Shape P deg.deg. χ deg. ID deg. deg. B. cereus AdoP; w.t. Ado, SO4 3UAW 1.20 [29] 4T3 229 30 −20 +103 4 B. cereus AdoP; w.t. Ino, SO4 3UAX 1.20 [29] 4TT3 226 20330 30−21 27+101 +167 3 − AdoP; T2 191 33 32 +178 3 − B. cereus D204N Ado, SO4 3UAY 1.40 [29] 4T3 2 229 28 −18 +102 UP, w.t. [52] 3T 195 30 30 178 V. cholerae Thd 4LZW 1.29 4 − − mutant 3T 203 30 28 +165 AdoP; 2 − 3T 193 33 33 +177 B. cereus D204N Ino, SO4 3UAZ 1.40 [29] 4T3 2 223 29 −21 − +99 3T 195 30 29 180 mutant 4 − − S. oneidensis UP, w.t. Urd, SO4 4R2W 1.60 [31] OT 266 20 2 +133 OT1 4 275 27 +2 −+94 OT 264 33 4 +125 OT1 4 277 28 +3 −+93 T 266 31 2 +122 V. cholera PNP; w.t. Ado 1VHW 1.54 [40] O 1 S. mansoni UP, w.t. Thd, SO4 4TXJ 1.66 [26] OT 1 267 29 −1 −+95 OT1 275 31 +3 +124 OT 4 276 26 +3 +93 OT 269 31 0 +130 2,2’-O- 4T 230 29 19 +111 3 − S. typhimurium UP, w.t. anhydrouridine, 3FWP 1.86 [53] 4T 234 32 19 +111 O − SO4 4T 231 30 19 +110 3 −

The data in Table3 show that, in all six complexes of thymidine with Vibrio cholerae UP (PDB 4LZW) [52], sulfate ions are absent, thymidine has an anti orientation (χ is in the range of 180◦), 4 2 − and the furanose rings adopt 3T or 3T conformations (P is 191–203◦). In the dimeric structure 4TXJ [26], all four crystallographically independent complexes of Shistosoma mansoni UP with thymidine contain the nucleoside in the immediate vicinity of the high-syn region (χ 4 1 is in the range of 122–130◦), and the furanose rings adopt the OT and OT conformations (P is in the range of 266–275◦). Sulfate ions are present in all active sites. Uridine adopts a nearly identical conformation in the active site of Shewanella oneidensis UP (PDB 4 4R2W) [31] in the presence of sulfate (χ = 133◦, P = 266◦, OT ). Therefore, thymidine and uridine adopt similar conformations in the presence of sulfate, i.e., the presence or the absence of the 20-OH group in the nucleoside is not a crucial factor that is responsible for the conformation. In Figure5, the pseudoratation angles of uridine or thymidine in the active sites of UP in the presence of sulfate ions are shown as yellow and, when sulfate ions are absent, they are shown as light pink. The conformation of 2,2’-O-anhydrouridine is high-syn (χ = 111◦) with P ~230◦. This conformation is observed in all three active sites in complexes with Salmonella typhimurium UP (PDB 3FWP) [53] and it is determined by the C2’–O–C2 covalent bond. The rigid structure of 2,2’-O-anhydrouridine is well accommodated by the active site of uridine phosphorylase. The constant Ki of this substrate analogue to UP is only eight times lower as compared to Km of Urd and 13 times lower than Km of dUrd [54]. Biomolecules 2020, 10, 552 11 of 14

3. Conclusions The conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases determined at 2 Å resolution or higher were analyzed. In all complexes with nucleoside phosphorylases, nucleosides adopt similar conformations. They are signiﬁcantly diﬀerent from those of free nucleosides. The pentofuranose rings of all nucleosides in complexes are located within the W region of the pseudorotation cycle. Some of them are near the energy barrier of the N S ↔ interconversion. The presence of sulfate (or phosphate) ions in the structures limits the range over which the conformations of the pentofuranose rings can be varied. These limitations are associated with the fact that sulfate (or phosphate) ions form additional hydrogen bonds with atoms of nucleosides in the complexes. Most of the nucleosides (both purine and pyrimidine) are in a high syn orientation (in the immediate vicinity of the barrier to syn anti transitions at about χ = 120 ). The syn orientation is ↔ ◦ energetically highly unfavorable for pyrimidine nucleosides in the free form. To sum up, nucleosides adopt sterically strained conformations in the complexes. An increase in the potential energy of nucleosides in complexes is comparable with the energy of barriers to the N S ↔ interconversion through the W region and the height of the barrier to the syn anti transition in free ↔ nucleosides. The binding of nucleosides in strained conformations is a characteristic feature of the enzyme–substrate complex formation for this group of enzymes and it plays an important role in the mechanism of enzymatic catalysis.

Funding: This work was supported by the Russian Science Foundation (project No. 16-14-00178). Acknowledgments: We are grateful to Zarubin Yu.P. for providing us with the program for calculating the parameters for pseudorotation within the pentofuranose cycle. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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