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.     

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production

trans-4-Hydroxy- l -proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l -proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ⁻¹·hr ⁻¹ in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.     

Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst

A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.     

L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化

L-脯氨酸-4-羟化酶(L-Proline-4-hydroxylase,P4H)是依赖α-酮戊二酸(α-KG)和Fe2+的双加氧酶成员之一,在反式-4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline,t-4Hyp)等重要手性化合物的生物合成中发挥关键作用。本研究构建了来源于Bradyrhizobium japonicum USDA 6的P4H重组大肠杆菌Escherichia coli BL21(DE3)/p ET-28b-p4h BJ,SDS-PAGE和酶活检测结果表明,该菌株具有表达可溶性P4H和催化合成t-4Hyp的能力。通过优化,确定了该重组菌全细胞催化合成t-4Hyp较优的反应体系和条件:10 m L p H 6.5 80 mmol/LMES缓冲液、9 mmol/L L-Pro,6 mmol/L L-抗坏血酸,6 mmol/Lα-KG,0.8 mmol/L Fe SO4·7H2O,反应温度为35℃;在20 g/L湿细胞的催化反应中,t-4Hyp的合成量达到34.86 mg/L,比优化前(17.53 mg/L)提高了98.86%。该工作为进一步利用P4H生物催化法合成t-4Hyp奠定了一定的技术基础。     

Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His₆-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX₂₄TX₁₃₁HX₁₀R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent K_m values were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparent K_m values for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAα-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.     

A Novel Whole-Cell Biocatalyst with NAD+ Regeneration for Production of Chiral Chemicals

Background

The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)⁺ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.

Methodology/Principal Findings

Simultaneous overexpression of an NAD⁺ dependent enzyme and an NAD⁺ regenerating enzyme (H₂O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD⁺-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.

Conclusions/Significance

A recombinant strain, in which an NAD⁺ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)⁺ for catalysis.     

Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures

Dilute salt solutions eluted peroxidase and hydroxyproline-rich glycoproteins (HRGP's) very rapidly (60 % within 10s) from the surface of intact tomato cells grown in suspension culture. Further purification of the HRGPs based on (a) their solubility in 10% trichloroacetic acid and (b) chromatography on carboxymethyl cellulose, gave two components (P1 and P2) rich in serine, tyrosine, lysine and arabinosylated hydroxyproline. The sum of the hydroxyproline arabinoside profiles of P1 and P2 approximated that of the wall. P1, unlike P2, was histidine-rich and also contained proline. Significantly, isodityrosine (IDT) was absent from P1 and P2 but present in cell wall hydrolysates where, the Hyp:IDT molar ratio was ca 15: 1. In cells 4 days after subculture, ³H-proline pulse-chase data indicated turnover of P1 and P2 presumably resulting from covalent attachment to the wall as neither P1 nor P2 appeared in the growth medium. At day four the cell mean generation time (MGT) was 4.6 days, the cell hydr oxyproline content was 0.7 % (w/w), the half lives of P1 and P2 were both ca 12 hr, and the combined CaCl₂ elutable P1 and P2 precursor pools contained ca 400 μg Hyp/g cells (dry weight). Calculated from the MGT and Hyp content, the cell demand was 44.μg Hyp/g cells (dry weight)/hr. The precursor pool size was therefore sufficient for 9 hours growth. However the pool turnover calculated from half life and pool size was 5.6 %/hr or 22.4μg Hyp/g cells (dry weight)/hr. Thus the supply of P1 and P2 precursors met > 50 % of the cell wall demand. Corroborative experiments showed that after depletion of the P1 and P2 pools by salt elution, washed cells resuspended in growth medium repleted the precursor pools at a rate corresponding to a synthesis of 43μg Hyp/g cells (dry weight)/hr, or 98 % of the demand. These data allow us to make the following suggestions: P1 and P2 represent monomeric extensin precursor subunits. Salt elution of P1 and P2 indicates their ionic binding by pectic carboxyl groups. The rapidity of elution indicates a high diffusivity of these extended rodlike macromolecules through the cell wall. This may imply a preferred orientation for P1 and P2 perpendicular rather than parallel to the plane of the wall. The lack of IDT in P1 and P2 implies that IDT forms in muro, possibly via peroxidase. We speculate that some of these IDT residues may crosslink an extensin precursor ‘tweft’ around a cellulose microfibrillar ‘twarp’. Such formation of heteromultimeric extensin interpenetrated by microfibrils would create a mechanically coupled extensin-cellulose network.     

2(S),3(S)-3-hydroxy-4-methyleneglutamic Acid from Gleditsia caspica

A new natural product, 2(S),3(S)-3-hydroxy-4-methyleneglutamic acid (G3) has been isolated from seeds of Gleditsia caspica. The structure has been established by chemical and spectroscopic methods. Catalytic reduction of G3 yields 2(S),4(S)-4-methylglutamic acid and a new amino acid, 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid. Ozonolysis of G3 followed by oxidation gives 2(S),3(R)-3-hydroxyaspartic acid. The S- (or l-) configurations at C₂ in G3 and in 2(S),3(S),4(S)-3-hydroxy-4-methyglutamic acid and the S-configurations at C₃ for G3 and 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid and at C₄ for 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid are inferred from the configurations at C₂ in 2(_S),4(_S)-4-methylglutamic acid and at C₂ and C₃ in 2(S),3(R)-3-hydroxyaspartic acid. The seeds also contain appreciable quantities of 2(S),3(S),4(R)-3-hydroxy-4-methylglutami c acid (G1) and 2(S),4(R)-4-methylglutamic acid.     

Dibenzocyclooctadiene lignans from Magnolia and Talauma (Magnoliaceae): Their absolute configuration ascertained by circular dichroism and X-ray crystallography and re-evaluation of previously published pyramidatin structures

Twelve pyramidatins, i.e., dibenzocyclooctadiene-type lignans, together with Machilin G, were isolated from the dichloromethane extracts of aerial material of Talauma gloriensis, Magnolia fraseri, and Magnolia pyramidata (Magnoliaceae). These lignans contain a highly oxidized 7,9′-epoxy-2,2′-cyclolignane skeleton. Their structures were established using NMR spectroscopy (1D and 2D experiments) and mass spectrometry. The absolute configurations of five pairs of atropisomers (S_a/R_a-pyramidatins) and two single atropisomers (S_a-pyramidatins) were determined by experimental and calculated circular dichroism (CD). In addition, the absolute configuration of (S_a)-3,3′,4,4′,5,5′-hexamethoxypyramidatin was confirmed using X-ray crystallography.Five pyramidatins, (R_a)-3,3′,4,4′,5,5′-hexamethoxypyramidatin, (R_a)-3,3′-dimethoxy-4,5:4′,5′-bis(methylenedioxy)pyramidatin, (S_a)-3,3′,4,5′-tetramethoxy-4,5-methylenedioxypyramidatin, (R_a)-3,3′,4,5′-tetramethoxy-4,5-methylenedioxypyramidatin, and (R_a)-3,3′,4,5-tetramethoxy-4′,5′-methylenedioxypyramidatin are reported herein for the first time. In the current dataset, NMR values are in accordance with the observed and calculated CD values. These values are herein reported with particular reference to previously described data of pyramidatins, which have to be revised.     

Complexes of N-thiophosphorylthioureas RNHC(S)NHP(S)(OiPr)2 (HL) (R = pyridin-2-yl, pyridin-3-yl, 6-amino-pyridin-2-yl) with copper(I): Crystal structures of Cu(PPh3)nL (n = 1, 2)

Reaction of the potassium salts of N-thiophosphorylated thioureas of common formula RNHC(S)NHP(S)(OiPr)₂ [R = pyridin-2-yl (HL^a), pyridin-3-yl (HL^b), 6-amino-pyridin-2-yl (HL^c)] with Cu(PPh₃)₃I in aqueous EtOH/CH₂Cl₂ leads to mononuclear [Cu(PPh₃)₂L^a,b-S,S′] (1, 2) and [Cu(PPh₃)L^c-S,S′] (3) complexes. Using copper(I) iodide instead of Cu(PPh₃)₃I, polynuclear complexes [Cu_n(L-S,S′)_n] (4-6) were obtained. The structures of these compounds were investigated by IR, ¹H, ³¹P{¹H} NMR spectroscopy, ES-MS and elemental analyses. The crystal structures of Cu(PPh₃)₂L^b (2) and Cu(PPh₃)L^c (3) were determined by single-crystal X-ray diffraction.     

Origin of the stability conferred upon collagen by fluorination

According to a prevailing theory, (2S,4R)-4-hydroxyproline (Hyp) residues stabilize the collagen triple helix via a stereoelectronic effect that preorganizes appropriate backbone torsion angles for triple-helix formation. This theory is consistent with the marked stability that results from replacing the hydroxyl group with the more electron-withdrawing fluoro group, as in (2S,4R)-4-fluoroproline (Flp). Nonetheless, the hyperstability of triple helices containing Flp has been attributed by others to the hydrophobic effect rather than a stereoelectronic effect. We tested this hypothesis by replacing Hyp with 4,4-difluoroproline (Dfp) in collagen-related peptides. Dfp retains the hydrophobicity of Flp, but lacks the ability of Flp to preorganize backbone torsion angles. Unlike Flp, Dfp does not endow triple helices with elevated stability, indicating that the hyperstability conferred by Flp is not due to the hydrophobic effect.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

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.     

Silver(I) derivatives with new functionalised acylpyrazolonates

Silver(I) acylpyrazolonate derivatives of formula [Ag(Q)(R₃P)]₂ and [Ag(Q)(R₃P)₂], (QH=1-phenyl-3-methyl-4-R′(CO)-pyrazol-5-one; Q^OH, R′=furane; Q^SH, R′=thiophene; R=Ph, Cy, o-tol), have been synthesised and characterised, both in the solid state and in solution. The derivatives [Ag(Q)(R₃P)]₂ contain dinuclear AgO₂NP units with the acylpyrazolonate coordinating in a bridging O,O′-Q-N fashion. The [Ag(Q)(R₃P)₂] are tetrahedral species, with the distortion from ideal geometry increasing with the bulk of the phosphine. The [Ag(Q)(R₃P)₂] derivatives are fluxional in chloroform solution when R₃P is sterically hindered (R=Cy or o-tol), dissociating partially to the [Ag(Q)(R₃P)] fragment and free R₃P. [Ag(Q^S)(Ph₃P)]₂ reacts with 1-methyl-2-mercaptoimidazole (Hmimt) affording the compound [Ag(Hmimt)(Ph₃P)(Q^S)] and [Ag(Q^O)(Ph₃P)]₂ reacts with 1-methyl-imidazole (Meim) affording the compound [Ag(Meim)(Ph₃P)(Q^O)], whereas [Ag(Q^S)(Ph₃P)]₂ reacts with 1,10-phenanthroline (phen), affording the compound [Ag(phen)(Ph₃P)](Q^S). Finally [Ag(Q^S)(Ph₃P)₂] reacts with phen producing the ionic species [Ag(phen)(Ph₃P)₂](Q^S).     

Fullerene‐based inhibitors of HIV‐1 protease

A series of Fmoc‐Phe(4‐aza‐C₆₀)‐OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4‐aza‐C₆₀)‐OH, is derived from the dipolar addition to C₆₀ of the Fmoc‐Nα‐protected azido amino acids derived from phenylalanine: Fmoc‐Phe(4‐aza‐C₆₀)‐Lys₃‐OH ( 1 ), Fmoc‐Phe(4‐aza‐C₆₀)‐Pro‐Hyp‐Lys‐OH ( 2 ), and Fmoc‐Phe(4‐aza‐C₆₀)‐Hyp‐Hyp‐Lys‐OH ( 3 ). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET‐based assay to evaluate the enzyme kinetics of HIV‐1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc‐Phe‐Pro‐Hyp‐Lys‐OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C₆₀‐based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The enzymatic synthesis of S-adenosyl-l-[2(n)-H]homocysteine

A rapid, efficient method is described for the enzymatic conversion of S-adenosyl-l-[2(n)-³H]methionine to S-adenosyl-l-[2(n)-³H]homocysteine. Partially purified glycine N-methyltransferase is used in the reaction which yields 98% conversion. The product is purified using high-pressure liquid chromatography and is concentrated by lyophilization. S-Adenosyl-l-[2(n)-³H]homocysteine synthesized by this method is an active substrate for S-adenosylhomocysteine (SAH) hydrolase. A novel assay procedure for SAH hydrolase is also described, in which unreacted S-adenosyl-l-[2(n)-³H]homocysteine is removed by adsorption to dextran-coated charcoal.     

Anti-AIDS agents 84. Synthesis and anti-human immunodeficiency virus (HIV) activity of 2′-monomethyl-4-methyl- and 1′-thia-4-methyl-(3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogs

In a continuing investigation into the pharmacophores and structure–activity relationship (SAR) of (3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) as a potent anti-HIV agent, 2′-monomethyl substituted 1′-oxa, 1′-thia, 1′-sulfoxide, and 1′-sulfone analogs were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Among them, 2′S-monomethyl-4-methyl DCK (5a)³ and 2′S-monomethyl-1′-thia-4-methyl DCK (7a) exhibited potent anti-HIV activity with EC₅₀ values of 40.2 and 39.1 nM and remarkable therapeutic indexes of 705 and 1000, respectively, which were better than those of the lead compound DCK in the same assay. In contrast, the corresponding isomeric 2′R-monomethyl-4-methyl DCK (6) and 2′R-monomethyl-1′-thia-4-methyl DCK (8) showed much weaker inhibitory activity against HIV-1 replication. Therefore, the bioassay results suggest that the spatial orientation of the 2′-methyl group in DCK analogs can have important effects on anti-HIV activity of this compound class.     

Fructose-1, 6-bisphosphate aldolase from rabbit muscle: Different catalytic behavior of the dihydroxyacetone phosphate binding sites at low temperature

The equivalence of the four dihydroxyacetone phosphate binding sites of aldolase was abolished by lowering the temperature. At pH 6.2 and ?13 2C, four binding sites were detected by gel filtration; two sites with a K_diss ?0.1 μm, and a second set of sites with a K_diss = 4 μm. The alteration of the binding was accompanied by the alteration of the catalytic activity. The low-affinity sites were incapable of catalyzing the cleavage of the (3S) CH bond of dihydroxyacetone phosphate, and form only the ketimine phosphate intermediate. The high-affinity sites were still able to cleave the (3S) CH bond of dihydroxyacetone phosphate; however, the eneamine phosphate intermediate formed was almost fully converted into the eneamine-aldehyde … phosphate intermediate, which was the prevailing species at the equilibrium. The mechanism of the half-of-the sites reactivity of aldolase at low temperature has been explained and the nonequivalence of sites in promoting catalysis has been utilized to dissect and characterize the individual partial reactions of the enzyme. In the course of these studies it has been shown that the rate of hydration-dehydration of dihydroxyacetone phosphate at ?24 °C was too slow to measure.     

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.     

Absorbance difference spectra of the successive redox states of the oxygen-evolving apparatus of photosynthesis

The spectra of the absorbance changes due to the turnover of the so-called S-states of the oxygen-evolving apparatus were determined. The changes were induced by a series of saturating flashes in dark-adapted Photosystem II preparations, isolated from spinach chloroplasts. The electron acceptor was 2,5-dichloro-p-benzoquinone. The fraction of System II centers involved in each S-state transition on each flash was calculated from the oscillation pattern of the 1 ms absorbance transient which accompanies oxygen release. The difference spectrum associated with each S-state transition was then calculated from the observed flash-induced difference spectra. The spectra were found to contain a contribution by electron transfer at the acceptor side, which oscillated during the flash series approximately with a periodicity of two and was apparently modulated to some extent by the redox state of the donor side. At the donor side, the S₀ → S₁, S₁ → S₂ and S₂ → S₃ transitions were all three accompanied by the same absorbance difference spectrum, attributed previously to an oxidation of Mn(III) to Mn(IV) (Dekker, J.P., Van Gorkom, H.J., Brok, M. and Ouwehand, L. (1984) Biochim. Biophys. Acta 764, 301–309). It is concluded that each of these S-state transitions involves the oxidation of an Mn(III) to Mn(IV). The spectrum and amplitude of the millisecond transient were in agreement with its assignment to the reduction of the oxidized secondary donor Z⁺ and the three Mn(IV) ions.