Two Novel Iboga-Type and an Oxindole Glucuronide Alkaloid from Tabernaemontana peduncularis Disclose Related Biosynthetic Pathways to Tabernaemontana divaricata.

Phytochemical investigation of the two Tabernaemontana species (Apocynaceae) T. peduncularis Wall. and T. divaricata (L.) R.Br. ex Roem. & Schult. indicated closely related biosynthetic pathways leading to lipophilic and hydrophilic alkaloids. In total, 18 specialized metabolites comprising indole-derived alkaloid aglycones, three oxindole-derived alkaloid glycosides, and two iridoid glucosides could be identified in the studied species. Among the alkaloids, the two Iboga-type alkaloids 3,7-coronaridine isoindolenine, coronaridine 3,4-iminium and a javaniside derivative bearing a glucuronic acid, named javanuronic acid, could be described by spectroscopic and spectrometric methods for the first time. A docking experiment using alpha-fold was performed to generate a protein model of the enzyme 7-deoxyloganetic acid glucosyl transferase. Performed bioassays exhibited a growth reduction of neonate Spodoptera littoralis larvae and reduced cell viability of HepG2 cells of the extracts containing Iboga alkaloids, whilst the javaniside derivatives containing hydrophilic fraction did not show any effects. These findings indicate a high flexibility in the formation of bioactive indole alkaloid aglycones by Tabernaemontana species and also evidence similar accumulation trends in both species as well as indicate that biosynthetic routes leading to oxindole alkaloids like javanisides are more widespread than reported. Furthermore, the incorporation of the three novel compounds into potential biosynthetic pathways is discussed.

MAPkinases regulate secondary metabolism, sexual development and light dependent cellulase regulation in Trichoderma reesei.

The filamentous fungus Trichoderma reesei is a prolific producer of plant cell wall degrading enzymes, which are regulated in response to diverse environmental signals for optimal adaptation, but also produces a wide array of secondary metabolites. Available carbon source and light are the strongest cues currently known to impact secreted enzyme levels and an interplay with regulation of secondary metabolism became increasingly obvious in recent years. While cellulase regulation is already known to be modulated by different mitogen activated protein kinase (MAPK) pathways, the relevance of the light signal, which is transmitted by this pathway in other fungi as well, is still unknown in T. reesei as are interconnections to secondary metabolism and chemical communication under mating conditions. Here we show that MAPkinases differentially influence cellulase regulation in light and darkness and that the Hog1 homologue TMK3, but not TMK1 or TMK2 are required for the chemotropic response to glucose in T. reesei. Additionally, MAPkinases regulate production of specific secondary metabolites including trichodimerol and bisorbibutenolid, a bioactive compound with cytostatic effect on cancer cells and deterrent effect on larvae, under conditions facilitating mating, which reflects a defect in chemical communication. Strains lacking either of the MAPkinases become female sterile, indicating the conservation of the role of MAPkinases in sexual fertility also in T. reesei. In summary, our findings substantiate the previously detected interconnection of cellulase regulation with regulation of secondary metabolism as well as the involvement of MAPkinases in light dependent gene regulation of cellulase and secondary metabolite genes in fungi.

New cytochalasans from an endophytic Xylaria species associated with Costa Rican Palicourea elata (Rubiaceae).

Four new leucine-derived cytochalasans, possessing a 5,6,5,8-ring (1) and a 5,6,11-ring core (2-4), were isolated from a cultivated endophytic fungus Xylaria sp. strain WH2D4 (Xylariaceae). This fungus was isolated from leaves of the neotropical tree species Palicourea elata (Sw.) Borhidi (Rubiaceae) collected in Costa Rica. The chemical structures were determined by employing IR, MS as well as 1D- and 2D-NMR experiments. The stereochemistry at C-15 of compound 4 was determined by quantum calculations. The isolated compounds did not affect germination and growth of Trichoderma reesei and the opportunistic human fungal pathogen T. longibrachiatum.

Phytochemistry Meets Geochemistry­Blumenol C Sulfate: A New Megastigmane Sulfate from Palicourea luxurians (Rubiaceae: Palicoureeae)

There is a previously neglected influence of geochemical conditions on plant phytochemistry. In particular, high concentrations of dissolved salts can affect their biosynthesis of natural products. Detoxification is most likely an important aspect for the plant, but additional natural products can also give it an expanded range of bioactivities. During the phytochemical analysis a Palicourea luxurians plant collected in a sulfate-rich environment (near the Río Sucio, Costa Rica) showed an interesting natural product in this regard. The structure of this compound was determined using spectroscopic and computational methods (NMR, MS, UV, IR, CD, optical rotation, quantum chemical calculations) and resulted in a megastigmane sulfate ester possessing a ß-ionone core structure, namely blumenol C sulfate (1, C13H22O5S). The levels of sulfur and sulfate ions in the leaves of the plant were determined using elemental analysis and compared to the corresponding levels in comparable plant leaves from a less sulfate-rich environments. The analyses show the leaves from which we isolated blumenol C sulfate (1) to contain 35% more sulfur and 80% more sulfate than the other samples. Antimicrobial and antioxidant activities of compound 1 were tested against Escherichia coli, E. coli ampR and Bacillus subtilis as well as measured using complementary in vitro FRAP and ATBS assays, respectively. These bioactivities are comparable to those determined for structurally related megastigmanes. The sulfur and sulfate content of the plant leaves from the sulfate-rich environment was significantly higher than that of the other plants. Against this background of salt stress, we discuss a possible biosynthesis of blumenol C sulfate (1). Furthermore, there appears to be no benefit for the plant in terms of extended bioactivities. Hence, the formation of blumenol C sulfate (1) probably primarily serves the plant detoxification process.

Yellow Twig (Nauclea orientalis) from Thailand: Strictosamide as the Key Alkaloid of This Plant Species

Comprehensive phytochemical examination from different perspectives using preparative and analytical chromatographic techniques combined with spectroscopic/spectrometric methods of the so-called "yellow twig" Nauclea orientalis (L.) L. (Rubiaceae) led to the identification of 13 tryptamine-derived (=monoterpene-indole) alkaloids. The identified alkaloids comprise strictosamide and four of its glucosidic derivatives, three oxindole derivatives, and five yellow-colored angustine-type aglycones. Qualitative and quantitative HPLC analyses showed the enrichment of strictosamide in all studied organs. Based on these results, we performed metabolomic analyses of monoterpene-indole alkaloids and made a 1H NMR in vitro monitoring of enzymatic deglucosylation of strictosamide. A comparison of the stability of strictosamide and its enantiomer vincoside lactam by theoretical calculations was also performed revealing a slightly higher stability of vincoside lactam. Additionally, we conducted two different anti-feedant assays of strictosamide using larvae of the polyphageous moth Spodoptera littoralis Boisduval. The obtained results indicate that generally two different biosynthetic pathways are most likely responsible for the overall alkaloid composition in this plant. Strictosamide is the key compound in the broader pathway and most likely the source of the identified angustine-type aglycones, which may contribute significantly to the yellow color of the wood. Its cross-organ accumulation makes it likely that strictosamide is not only important as a reservoir for the further biosynthesis, but also acts in the plants' defense strategy.

Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase.

The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is ∼104-fold. Cooperativity with the catalytic acid adds ≥102-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

Dissecting Metabolism of Leaf Nodules in Ardisia crenata and Psychotria punctata.

Root-microbe interaction and its specialized root nodule structures and functions are well studied. In contrast, leaf nodules harboring microbial endophytes in special glandular leaf structures have only recently gained increased interest as plant-microbe phyllosphere interactions. Here, we applied a comprehensive metabolomics platform in combination with natural product isolation and characterization to dissect leaf and leaf nodule metabolism and functions in Ardisia crenata (Primulaceae) and Psychotria punctata (Rubiaceae). The results indicate that abiotic stress resilience plays an important part within the leaf nodule symbiosis of both species. Both species showed metabolic signatures of enhanced nitrogen assimilation/dissimilation pattern and increased polyamine levels in nodules compared to leaf lamina tissue potentially involved in senescence processes and photosynthesis. Multiple links to cytokinin and REDOX-active pathways were found. Our results further demonstrate that secondary metabolite production by endophytes is a key feature of this symbiotic system. Multiple anhydromuropeptides (AhMP) and their derivatives were identified as highly characteristic biomarkers for nodulation within both species. A novel epicatechin derivative was structurally elucidated with NMR and shown to be enriched within the leaf nodules of A. crenata. This enrichment within nodulated tissues was also observed for catechin and other flavonoids indicating that flavonoid metabolism may play an important role for leaf nodule symbiosis of A. crenata. In contrast, pavettamine was only detected in P. punctata and showed no nodule specific enrichment but a developmental effect. Further natural products were detected, including three putative unknown depsipeptide structures in A. crenata leaf nodules. The analysis presents a first metabolomics reference data set for the intimate interaction of microbes and plants in leaf nodules, reveals novel metabolic processes of plant-microbe interaction as well as the potential of natural product discovery in these systems.

Highly Aromatic Flavan-3-ol Derivatives from Palaeotropical Artocarpus lacucha Buch.-Ham Possess Radical Scavenging and Antiproliferative Properties.

Phytochemical investigation of leaves and stembark of Artocarpus lacucha collected in Thailand resulted in three yet undescribed isomeric flavan-3-ol derivatives (1-3), the four known compounds gambircatechol (4), (+)-catechin (5), (+)-afzelechin (6) and the stilbene oxyresveratrol (7). Compounds 1 to 3 feature 6/6/5/6/5/6 core structures. All structures were deduced by NMR and MS, while density functional theory (DFT) calculations on B3LYP theory level were performed of compounds 1 to 3 to support the stereochemistry in positions 2 and 3 in the C-ring. Possible biosynthetic pathways leading to 4 are discussed. The DPPH assay revealed high radical scavenging activities for 1 (EC50 = 9.4 ± 1.0 µmol mL-1), 2 (12.2 ± 1.1), 3 (10.0 ± 1.5) and 4 (19.0 ± 2.6), remarkably lower than ascorbic acid (EC50 = 34.9) and α-tocopherol (EC50 = 48.6). A cytotoxicity assay revealed moderate but consistent antiproliferative properties of 1 in CH1/PA-1 (ovarian teratocarcinoma) and SW480 (colon carcinoma) cells, with IC50 values of 25 ± 6 and 34 ± 4 µM, respectively, whereas effects in A549 (non-small cell lung cancer) cells were rather negligible. The performed DCFH-DA assay of 1 in the former cell lines confirmed potent antioxidative effects even in the cellular environment.

Chemical constituents of Clausena lenis.

Phytochemical examination of Clausena lenis Drake (Rutaceae), collected in Thailand, led to the isolation of seven coumarins, four furoquinolines, two amides, and one flavonoid glycoside. Four of these compounds, one coumarine derivative named as gravelliferone A (3), two furoquinoline derivatives (kokusagenin A (8) and B (9)) and one amide, clausenalansamide H (13), are reported for the first time. Compound 3 was isolated from the root bark, compound 8 from the stem bark and compounds 9 and 13 from the leaves. The molecular structures of all isolated compounds were established by means of NMR experiments combined with mass spectrometry. Preliminary tests of the lipophilic stem bark extract against various human pathogenic bacteria strains revealed promising effects against Staphylococcus aureus ATCC 43300.

Nuclear Magnetic Relaxation Mapping of Spin Relaxation in Electrically Stressed Glycerol.

This work discusses nuclear magnetic relaxation effects in glycerol subject to a strong electric field. The methods used are 1.5 T magnetic resonance imaging (MRI), referenced by 9.4 T nuclear magnetic resonance (NMR). While MRI allows a glycerol probe to be sampled with a high voltage (HV) of 16 kV applied to the probe, NMR provides precise molecular data from the sample, but the sample cannot be tested under HV. Using MRI, the recording of magnetic relaxation times was possible while HV was applied to the glycerol. NMR spectroscopy was used to confirm that MRI provides a reasonably accurate estimation of temperature. The applied HV was observed to have a negligible effect on the spin-lattice relaxation time T 1, which represents the energy release to the thermal bath or system enthalpy. In contrast to that, the spin-spin relaxation time T 2, which does represent the local entropy of the system, shows a lower response to temperature while the liquid is electrically stressed. These observations point toward a proton population in electrically stressed glycerol that is more mobile than that found in the bulk, an observation that is in agreement with previously published results for water.

Alkaloid and iridoid glucosides from Palicourea luxurians (Rubiaceae: Palicoureeae) indicate tryptamine- and tryptophan-iridoid alkaloid formation apart the strictosidine pathway.

The first phytochemical examination of extracts from leaves and stem bark of Palicourea luxurians (Rusby) Borhidi yielded two undescribed and one known alstrostine derivative together with the oxindole alkaloid javaniside as well as with 5α-carboxystrictosidine. Additionally, five iridoids and four secologanin derived isolation artifacts have been isolated. Lack of strictosidine and its follow-up metabolization products suggested that the Pictet-Spenglerase in P. luxurians does barely or not catalyze the formation of strictosidine. Against this background the biosynthesis of javaniside and 5α-carboxystrictosidine is discussed with regard to possible reaction mechanisms. Similarly, P. luxurians used an independent biosynthetic pathway to produce alstrostine type structures from secologanin and tryptamine in a 2:1 ratio. The structure of isoalstrostine A, which was isolated for the first time, allowed the refinement of a previously reported pathway to the alstrostine type carbon skeleton as well as to some follow-up metabolization products. In spite of various biosynthetic pathways incorporating secologanin to gain different types of tryptophan- and tryptamine-iridoid alkaloids, P. luxurians accumulates this compound as well a couple of further metabolized iridoids deriving from loganin and secologanin.

The role of PKAc1 in gene regulation and trichodimerol production in Trichoderma reesei.

BACKGROUND: Trichoderma reesei represents a model system for investigation of plant cell wall degradation and its connection to light response. The cyclic adenosine monophosphate pathway (cAMP pathway) plays an important role in both physiological outputs, being crucial for regulation of photoreceptor function as well as for cellulase regulation on different carbon sources. Phosphorylation of photoreceptors and of the carbon catabolite repressor CRE1 was shown in ascomycetes, indicating a relevance of protein kinase A in regulation of the target genes of these transcription factors as well as an impact on regulation of induction specific genes. Moreover, the cAMP pathway impacts growth and development. RESULTS: Here, we investigated gene regulation by the catalytic subunit of protein kinase A (PKAc1) upon growth on cellulose. We found distinct gene sets for regulation upon growth in light and darkness with an overlap of only 13 genes. PKAc1 regulates metabolic genes as well as transport and defense functions. The overlap of gene regulation by PKAc1 with the genes representing the cAMP dependent regulatory output of the photoreceptor ENV1 indicates an involvement of PKA in this pathway, which counteracts its effects by contrasting regulation. Moreover, we found considerable overlap with the gene sets regulated under cellulase inducing conditions and by the carbon catabolite repressor CRE1. Our analysis also showed that PKAc1 regulates the genes of the SOR cluster associated with the biosynthesis of sorbicillinoids. The homologue of gin4, encoding a CAMK type kinase, which is regulated by PKAc1, CRE1 and YPR2 showed a moderate impact on trichodimerol production. We isolated trichodimerol as representative sorbicillin compound and established a method for its quantification in large sample sets using high performance thin layer chromatography (HPTLC), which can be broadly applied for secondary metabolite screening of mutants or different growth conditions. Due to the high expression levels of the SOR cluster under conditions of sexual development we crosschecked the relevance of PKAc1 under these conditions. We could show that PKAc1 impacts biosynthesis of trichodimerol in axenic growth and upon mating. CONCLUSIONS: We conclude that PKAc1 is involved in light dependent regulation of plant cell wall degradation, including carbon catabolite repression as well as secondary metabolism and development in T. reesei.

Reversible DNA compaction induced by partial intercalation of 16-Ph-16 gemini surfactants: evidence of triple helix formation.

The interaction between calf thymus DNA and the gemini surfactants N,N'-[α,ω-phenylenebis(methylene)bis [N,N'-dimethyl-N-(1-hexadecyl)]-ammonium dibromide], p-16-Ph-16 (α = 1, ω = 3) and m-16-Ph-16 (α = 1, ω = 2), has been investigated via circular dichroism, fluorescence and UV-vis spectroscopy, zeta potential, dynamic light scattering, and AFM microscopy. Measurements were carried out in aqueous media at different molar ratios, R = (C16-Ph-16)/CDNA and C16-Ph-16 always below the critical micellar concentration (CMC) of the surfactant. Under these conditions, DNA undergoes two reversible conformational changes, compaction and decompaction, due to interaction with the surfactant molecules at low and high molar ratios, respectively. The extent of such conformational changes is correlated with both the degree of surfactant partial intercalation, and the size and charge of the surfactant aggregates formed, in each case. Comparison of the results shows that the para-form of the surfactant intercalates into the DNA to a major extent; therefore, the compaction/decompaction processes are more effective. Among these, the structure of the resulting 16-Ph-16/DNA decompacted complex is worthy of note. For the first time it can be demonstrated that the partial intercalation of the 16-Ph-16 gemini surfactants induces the formation of triplex DNA-like structures at a high R ratio.

Chemodiversity of tryptamine-derived alkaloids in six Costa Rican Palicourea species (Rubiaceae-Palicoureeae).

We report 14 harmala and tryptamine-iridoid alkaloids with various tri-, tetra- and pentacyclic cores from leaves and stem bark of six species of the large and complex neotropical genus Palicourea. Among them is the previously undescribed compound deoxostrictosamide which is related to strictosamide, a key intermediate in camptothecin biosynthesis. In addition, we describe the occurrence of 1,2,3,4-tetrahydronorharman-1-one for the first time within Rubiaceae and ophiorine A and B, two alkaloids with an unusual core bearing a betaine function and a zwitterion as new for the genus. Although the other compounds are already known from other species, their degree of structural diversity highlights the remarkable biosynthetic capabilities of the genus Palicourea. Furthermore, the present paper provides additional support for the hypothesis that tryptamine-iridoid alkaloids represent a distinct chemosystematic feature for the genus Palicourea.

Binding pattern of intermediate UDP-4-keto-xylose to human UDP-xylose synthase: Synthesis and STD NMR of model keto-saccharides.

Human UDP-xylose synthase (hUXS1) exclusively converts UDP-glucuronic acid to UDP-xylose via intermediate UDP-4-keto-xylose (UDP-Xyl-4O). Synthesis of model compounds like methyl-4-keto-xylose (Me-Xyl-4O) is reported to investigate the binding pattern thereof to hUXS1. Hence, selective oxidation of the desired hydroxyl function required employment of protecting group chemistry. Solution behavior of synthesized keto-saccharides was studied without enzyme via 1H and 13C NMR spectroscopy with respect to existent forms in deuterated potassium phosphate buffer. Keto-enol tautomerism was observed for all investigated keto-saccharides, while gem-diol hydrate forms were only observed for 4-keto-xylose derivatives. Saturation transfer difference (STD) NMR was used to study binding of synthesized keto-gylcosides to wild type hUXS1. Resulting epitope maps were correlated to earlier published molecular modeling studies of UDP-Xyl-4O. STD NMR results of Me-Xyl-4O are in good agreement with simulations of the intermediate UDP-Xyl-4O indicating a strong interaction of proton H3 with the enzyme, potentially caused by active site residue Ala79. In contrast, pyranoside binding pattern studies of methyl uronic acids showed some differences compared to previously published STD NMR results of UDP-glycosides. In general, obtained results can contribute to a better understanding in binding of UDP-glycosides to other UXS enzyme family members, which have high structural similarities in the active site.

Synthesis, characterization, and antioxidant activity of Zn2+ and Cu2+ coordinated polyhydroxychalcone complexes.

ABSTRACT: Four new metal complexes [Cu(ISO)2], [Cu(BUT)2] and [Zn(ISO)2], [Zn(BUT)2] of the polyhydroxychalcones (isoliquiritigenin and butein) are synthesized, structurally characterized and their antioxidant activity is investigated. The formation of the complexes [Cu(ISO)2] and [Zn(ISO)2] is followed by Job's plot using NMR titration. The resulting compounds are characterized by mass spectrometry, IR spectroscopy, and elemental analysis. Studies on the radical scavenging activity are performed using DPPH as substrate. The results showed that the antioxidant activities of isoliquiritigenin and butein are enhanced after binding to copper or zinc.

Striking Diversification of Exudate Profiles in Selected Primula Lineages.

In continuation of previous studies on glandular exudates of Primula, we analyzed eleven so far unstudied species and several populations for exudate composition. Unsubstituted flavone and unusually substituted flavones, normally predominant in Primula exudates, were not detected in all of the analyzed samples. Instead, some species exhibited regular substituted flavonoids, and in some cases, no flavonoids could be detected at all. The detection of a diterpene (1) in P. minima exudates is new to Primula. On basis of MS and NMR, 1 was structurally characterized as ent-kaur-16-en-19-oic acid. Comparative profiling of exudates as performed by HPLC and TLC against authentic markers indicated further the presence of the benzoquinone primin and derivatives in some exudates. Thus, exudates of newly studied species contrast markedly with those analyzed so far. The significance of observed exudate diversification is discussed in view of the phylogeny of derived lineages in European alpine regions.

Phytochemical meanings of tetrahydro-ß-carboline moiety in strictosidine derivatives.

Synthesis of 13 different tetrahydro-ß-carbolines (THBC) was accomplished by applying the Pictet-Spengler reaction with seven aldehydes, which have been coupled with tryptamine (6) and l-tryptophan methyl ester (7), respectively. The resulting products represent analogues of strictosidine (1) and carboxystrictosidine (5). They were investigated with respect to possible effects on herbivores in feeding bioassays upon the generalist Spodoptera littoralis. Maximum inhibition averages were 42% after four and 46% after six days for the most effective product (19) at 1000ppm. Additionally, the frass of this particular bioassay was investigated via HPLC-UV for THBC digestion. All synthesized THBCs were also tested for their radical scavenger activity by monitoring their interaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds 16-20, 24 and 25 exhibited radical scavenging activity, ranging from 50% to 74% compared to that of α-tocopherol. All results were discussed with respect to possible contributions of tetrahydro-ß-carboline moieties in bioactivities of strictosidine (1) and its biodegradation products.

Diastereoselective Synthesis of Glycosyl Phosphates by Using a Phosphorylase-Phosphatase Combination Catalyst.

Sugar phosphates play an important role in metabolism and signaling, but also as constituents of macromolecular structures. Selective phosphorylation of sugars is chemically difficult, particularly at the anomeric center. We report phosphatase-catalyzed diastereoselective "anomeric" phosphorylation of various aldose substrates with α-D-glucose 1-phosphate, derived from phosphorylase-catalyzed conversion of sucrose and inorganic phosphate, as the phosphoryl donor. Simultaneous and sequential two-step transformations by the phosphorylase-phosphatase combination catalyst yielded glycosyl phosphates of defined anomeric configuration in yields of up to 70 % based on the phosphate applied to the reaction. An efficient enzyme-assisted purification of the glycosyl phosphate products from reaction mixtures was established.

Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed 4C1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp196, Glu237 and Asp295) and from Arg137. Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe52, a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe52 by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp196) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.