Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈ and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈ and (SP)₃₂ modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFP and (SP)₃₂‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈ module and AGP‐styled (SP)₃₂ module is proposed.     

Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition

Key message

Cell growth medium composition has profound impacts on the O -glycosylation of a “designer” arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein.

Abstract

Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)₃₂ tag (32 tandem repeats of “Ser-Pro” motif). The (SP)₃₂ tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)₃₂ tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

     

Hydroxyproline-O-glycosylated peptide tags enhance recombinant protein yields in tobacco transient expression

Plant transient expression provides a rapid production platform for recombinant proteins but is linked with low protein yields. To test if plant-specific hydroxyproline (Hyp)-O-glycosylated peptide tags attached to a target protein can improve overall yields of recombinant protein transiently expressed in Nicotiana benthamiana, enhanced green fluorescence protein (EGFP) was expressed as a fusion with 5 or 32 tandem repeats of a serine–proline motif, designated (SP)₅ or (SP)₃₂, which is known to direct extensive Hyp-O-glycosylation in plants. EGFP containing the (SP)_n motif showed enhanced yields in the order as follows: EGFP < EGFP-(SP)₅ ≪ (SP)₅-EGFP < (SP)₃₂-EGFP. The EGFP equivalent yield of (SP)₃₂-EGFP was up to 16-fold greater than that of the EGFP control. In addition, both fully glycosylated (SP)₃₂-EGFP (∼115 kDa) and partially glycosylated (SP)₃₂-EGFP (∼40 kDa) were detected in protein extracts of N. benthamiana. These two types of glycoforms were completely segregated between media and cells in tobacco BY-2 cell cultures.     

High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

Low‐yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline‐O‐glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY‐2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of ”Ser‐Pro“ dipeptide, or (SP)₃₂, to study cell growth and protein secretion, culture scale‐up, and establishment of perfusion cultures for continuous production. The BY‐2 cells accumulated low levels of cell biomass (～7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)₃₂‐tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY‐2 cells cultured in a 5‐L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day^?1, generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures.     

Localization of components of the oxidative cross-linking of glycoproteins and of callose synthesis in papillae formed during the interaction between non-pathogenic strains of Xanthomonas campestris and French bean mesophyll cells

Immunogold labelling was used to probe the responses of mesophyll cells in French bean ( Phaseolus vulgaris L.) to an hrpA mutant of Xanthomonas campestris pv. vesicatoria and a saprophytic strain of X.c. The non-pathogenic strains both caused localized alterations to the plant cell wall and formation of large papillae in adjacent cells. Immunocytochemistry showed the co-localization, in the cell wall and paramural deposits, of an M _r 42 000 proline-rich glycoprotein with chitin-binding activity (CBPRP) and the enzyme responsible for its immobilization, an M _r 46 000 peroxidase. The CBPRP appeared to lose antigenicity after cross-linking, and, unlike the peroxidase, was not detected consistently in the extracellular matrix that encapsulated bacteria onto the plant cell wall. The peroxidase may have a dual function in both the generation and utilization of H₂O₂ for cross-linking of proteins and phenolics during the construction of papillae. A burst of H₂O₂ was detected 1–5 h after inoculation at reaction sites by histochemical staining with cerium chloride. Progressive expansion of papillae and cell-wall alterations was, however, not associated with the maintenance of high levels of H₂O₂. Co-localization of callose and an M _r 65 000 polypeptide component of callose synthase was also demonstrated. Synthesis of callose appeared so rapid that the enzyme became embedded in the polysaccharide so that both were detected as integral to the developing papilla. Localized alterations to the cell wall and deposition of papillae were found to involve co-ordinated synthetic and oxidative activities at microsites within responding cells, without activation of the hypersensitive reaction.     

Flax rhamnogalacturonan lyases: phylogeny,differential expression and modeling of protein structure

Rhamnogalacturonan lyases (RGLs; EC 4.2.2.23) degrade the rhamnogalacturonan I (RG‐I) backbone of pectins present in the plant cell wall. These enzymes belong to polysaccharide lyase family 4, members of which are mainly from plants and plant pathogens. RGLs are investigated, as a rule, as pathogen ‘weapons’ for plant cell wall degradation and subsequent infection. Despite the presence of genes annotated as RGLs in plant genomes and the presence of substrates for enzyme activity in plant cells, evidence supporting the involvement of this enzyme in certain processes is limited. The differential expression of some RGL genes in flax (Linum usitatissimum L.) tissues, revealed in our previous work, prompted us to carry out a total revision (phylogenetic analysis, analysis of expression and protein structure modeling) of all the sequences of flax predicted as coding for RGLs. Comparison of the expressions of LusRGL in various tissues of flax stem revealed that LusRGLs belong to distinct phylogenetic clades, which correspond to two co‐expression groups. One of these groups comprised LusRGL6‐A and LusRGL6‐B genes and was specifically upregulated in flax fibers during deposition of the tertiary cell wall, which has complex RG‐I as a key noncellulosic component. The results of homology modeling and docking demonstrated that the topology of the LusRGL6‐A catalytic site allowed binding to the RG‐I ligand. These findings lead us to suggest the presence of RGL activity in planta and the involvement of special isoforms of RGLs in the modification of RG‐I of the tertiary cell wall in plant fibers.     

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulose production

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M_O, and for carbon source, M_C, are 0.85 mmol O₂/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y_X/O = 32.3 g biomass/mol O₂, Y_X/C = 1.1 g biomass/g C or Y_X/C = 0.44 g biomass/g hexose, Y_X/N = 12.5 g biomass/g nitrogen for the cell growth stage, and Y_X/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ～ 0.028 hr^?1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.     

Identification of three potent hydroxyproline O‐galactosyltransferases in Arabidopsis

Arabinogalactan proteins (AGPs) are plant‐specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O‐galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O‐galactosyltransferases, such that their disruption produces phenocopies of AGP‐deficient mutants, remain to be identified. Here, we purified and identified three potent Hyp O‐galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss‐of‐function analysis indicated that approximately 90% of the endogenous Hyp O‐galactosylation activity is attributable to these three enzymes. AGP14 expressed in the triple mutant migrated much faster on SDS‐PAGE than when expressed in wild‐type, confirming a considerable decrease in levels of glycosylation of AGPs in the mutant. Loss‐of‐function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp‐linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell‐to‐cell communication during plant growth and development.     

Alterations of iron distribution in Arabidopsis tissues infected by Dickeya dadantii

Dickeya dadantii is a plant‐pathogenic enterobacterium responsible for plant soft rot disease in a wide range of hosts, including the model plant Arabidopsis thaliana. Iron distribution in infected A. thaliana was investigated at the cellular scale using the Perls'–diaminobenzidine–H₂O₂ (PDH) method. Iron visualization during infection reveals a loss of iron from cellular compartments and plant cell walls. During symptom progression, two distinct zones are clearly visible: a macerated zone displaying weak iron content and a healthy zone displaying strong iron content. Immunolabelling of cell wall methylated pectin shows that pectin degradation is correlated with iron release from cell walls, indicating a strong relationship between cell wall integrity and iron in plant tissues. Using a D. dadantii lipopolysaccharide antibody, we show that bacteria are restricted to the infected tissue, and that they accumulate iron in planta. In conclusion, weak iron content is strictly correlated with bacterial cell localization in the infected tissues, indicating a crucial role of this element during the interaction. This is the first report of iron localization at the cellular level during a plant–microbe interaction and shows that PDH is a method of choice in this type of investigation.     

Chassis engineering of Escherichia coli for trans-4-hydroxy-l-proline production

Microbial production of trans-4-hydroxy-l -proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l -proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l⁻¹ Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l -proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb_(L)-DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l⁻¹, 87.4% and 1.38 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.     

Expression of the Human Milk Protein sCD14 in Tobacco Plant Cell Culture

The human milk protein sCD14 was expressed in tobacco plant cell cultures. Tobacco cells were transformed with a modified cd14 cDNA minus the GPI-tail and either the native human signal peptide (SP) or a plant SP, under the control of the CaMV-35S promoter. Transformants were screened using PCR and Southern blot analysis. The functionality of the inserted cDNA was checked by northern blot analysis for the presence of recombinant sCD14 mRNA. The detection of the protein has been observed by western blot analysis at an estimated level of 5 μg l⁻¹ in a non-soluble fraction of the culture medium.     

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk_on) and desorption (k_off) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.     

Impact of elevated ozone concentration on growth,physiology, and yield of wheat (Triticum aestivum L.): a meta‐analysis

We quantitatively evaluated the effects of elevated concentration of ozone (O₃) on growth, leaf chemistry, gas exchange, grain yield, and grain quality relative to carbon‐filtered air (CF) by means of meta‐analysis of published data. Our database consisted of 53 peer‐reviewed studies published between 1980 and 2007, taking into account wheat type, O₃ fumigation method, rooting environment, O₃ concentration ([O₃]), developmental stage, and additional treatments such as drought and elevated carbon dioxide concentration ([CO₂]). The results suggested that elevated [O₃] decreased wheat grain yield by 29% (CI: 24–34%) and aboveground biomass by 18% (CI: 13–24%), where CI is the 95% confidence interval. Even in studies where the [O₃] range was between 31 and 59 ppb (average 43 ppb), there was a significant decrease in the grain yield (18%) and biomass (16%) relative to CF. Despite the increase in the grain protein content (6.8%), elevated [O₃] significantly decreased the grain protein yield (?18%). Relative to CF, elevated [O₃] significantly decreased photosynthetic rates (?20%), Rubisco activity (?19%), stomatal conductance (?22%), and chlorophyll content (?40%). For the whole plant, rising [O₃] induced a larger decrease in belowground (?27%) biomass than in aboveground (?18%) biomass. There was no significant response difference between spring wheat and winter wheat. Wheat grown in the field showed larger decreases in leaf photosynthesis parameters than wheat grown in < 5 L pots. Open‐top chamber fumigation induced a larger reduction than indoor growth chambers, when plants were exposed to elevated [O₃]. The detrimental effect was progressively greater as the average daily [O₃] increased, with very few exceptions. The impact of O₃ increased with developmental stages, with the largest detrimental impact during grain filling. Both drought and elevated [CO₂] significantly ameliorated the detrimental effects of elevated [O₃], which could be explained by a significant decrease in O₃ uptake resulting from decreased stomatal conductance.     

A gymnosperm extensin contains the serine-tetrahydroxyproline motif

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp₄ motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp₄ motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp₄ motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp₄, in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp₃-Thr-Hyp-Tyr, Ser-Hyp₄-Lys, and (Ala-Hyp)_n repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp₄ is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.     

A Highly N-Glycosylated Chitin Deacetylase Derived from a Novel Strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2–4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1–10 mM Co²⁺ and strongly inhibited by 10 mM Cu²⁺. It required at least two GlcNAc residues for catalysis. When (GlcNAc)₆ was used as substrate, K _m and V _max were determined to be 1.1 mM and 54.6 μmol min^?1 respectively.     

Candida albicans mycelial wall structure: supramolecular complexes released by Zymolyase,chitinase and β-mercaptoethanol

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118–1125]. Zymolyase, chitinase and -mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (>180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.     

Prototropic tautomerism and basic molecular principles of hypoxanthine mutagenicity: an exhaustive quantum-chemical analysis

The molecular structures, relative stability order, and dipole moments of a complete family of 21 planar hypoxanthine (Hyp) prototropic molecular–zwitterionic tautomers including ylidic forms were computationally investigated at the MP2/6–311++G(2df,pd)//B3LYP/6–311++G(d,p) level of theory in vacuum and in three different surrounding environments: continuum with a low dielectric constant (??=?4) corresponding to a hydrophobic interface of protein–nucleic acid interactions, dimethylsulfoxide (DMSO), and water. The keto-N1HN7H tautomer was established to be the global minimum in vacuum and in continuum with ??=?4, while Hyp molecule exists as a mixture of the keto-N1HN9H and keto-N1HN7H tautomers in approximately equal amounts in DMSO and in water at T?=?298.15?K. We found out that neither intramolecular tautomerization by single proton transfer in the Hyp base, nor intermolecular tautomerization by double proton transfer in the most energetically favorable Hyp·Hyp homodimer (symmetry C _2h ), stabilized by two equivalent N1H…O6 H-bonds, induces the formation of the enol tautomer (marked with an asterisk) of Hyp with cis-oriented O6H hydroxyl group relative to neighboring N1C6 bond. We first discovered a new scenario of the keto–enol tautomerization of Hyp?·?Hyp homodimer (C _2h ) via zwitterionic near-orthogonal transition state (TS), stabilized by N1⁺H…N1^? and O6⁺H…N1^? H-bonds, to heterodimer Hyp^??·?Hyp (C _s ), stabilized by O6H…O6 and N1H…N1 H-bonds. We first showed that Hyp^??·?Thy mispair (C _s ), stabilized by O6H…O4, N3H…N1, and C2H…O2 H-bonds, mimicking Watson–Crick base pairing, converts to the wobble Hyp?·?Thy base pair (C _s ), stabilized by N3H…O6 and N1H…O2 H-bonds, via high- and low-energy TSs and intermediate Hyp?·?Thy^?, stabilized by O4H…O6, N1H…N3, and C2H…O2 H-bonds. The most energetically favorable TS is the zwitterionic pair Hyp⁺?·?Thy^? (C _s ), stabilized by O6⁺H…O4^?, O6⁺H…N3^?, N1⁺H…N3^?, and N1⁺H…O2^? H-bonds. The authors expressed and substantiated the hypothesis, that the keto tautomer of Hyp is a mutagenic compound, while enol tautomer Hyp^? does not possess mutagenic properties. The lifetime of the nonmutagenic tautomer Hyp^? exceeds by many orders the time needed to complete a round of DNA replication in the cell. For the first time purine–purine planar H-bonded mispairs containing Hyp in the anti-orientation with respect to the sugar moiety – Hyp?·?Ade _syn , Hyp?·?Gua^? _syn , and Hyp?·?Gua _syn , that closely resembles the geometry of the Watson–Crick base pairs, have been suggested as the source of transversions. An influence of the surrounding environment (??=?4) on the stability of studied complexes and corresponding TSs was estimated by means of the conductor-like polarizable continuum model. Electron-topological, structural, vibrational, and energetic characterictics of all conventional and nonconventional H-bonds in the investigated structures are presented. Presented data are key to understanding elementary molecular mechanisms of mutagenic action of Hyp as a product of the adenine deamination in DNA.