Colorimetric detection of alkaline phosphatase activity based on pyridoxal phosphate-induced chromatic switch of polydiacetylene nano-liposomes.

A colorimetric assay based on polydiacetylenes (PDA) nano-liposomes is reported for facile and sensitive detection of alkaline phosphatase (ALP) activity. The critical basis of this method is that the interaction of pyridoxal phosphate (PLP) with nitrogenous group functionalized PDA nano-liposomes induces distinct blue-to-red color changes of PDA nano-liposomes. In the presence of ALP, as a nature substrate, PLP is enzymatically hydrolyzed to form pyridoxal, which cannot interact with PDA nano-liposomes. As a result, the concentration of PLP is reduced and the color change of PDA nano-liposomes is retarded, which is associated with ALP level. Under optimal conditions, the proposed method showed good linear relationship with ALP activity in the range 10-200 U/L with a limit of detection of 2.8 U/L. The detection process could be vividly observed with the naked eye. Additional attempts by using the method for the evaluation of inhibitor efficiency were also achieved with satisfying results. The method was further challenged with real human serum samples, showing consistent results when compared with a commercial standard assay kit. Such simple and easy-to-use approach may provide a new alternative for clinical and biological detection of ALP.

Chitosan crosslinking with pyridoxal 5-phosphate vitamer toward biocompatible hydrogels for in vivo applications.

Vitamin B6 is an essential micronutrient in the mammalian diet, with role of coenzyme and synergistic effect with some antibiotics and antitumor drugs. Based on these, we hypothesized that its use for the preparation of hydrogels can yield multifunctional biomaterials suitable for in vivo applications. To this aim, chitosan was reacted with the active form of vitamin B6, pyridoxal 5-phosphate, via acid condensation, when clear hydrogels were obtained. Their investigation by structural characterization methods proved that the hydrogelation was a consequence of both covalent imine formation and physical interactions. The novel hydrogels had microporous morphology and showed shrinking effect in phosphate buffer, indicating good shape preservation and slow dissolution in in vivo environment. Their enzymatic biodegradation could be controlled by the imination degree, varying from 40 to 61% in 21 days. They demonstrated very good in vitro cytocompatibility on normal human dermal fibroblasts cells and no harmful effect on experimental mice, confirming their safely use for in vivo application.

Hierarchical Surface Architecture of Hemodialysis Membranes for Eliminating Homocysteine Based on the Multifunctional Role of Pyridoxal 5'-phosphate.

Patients with end-stage renal disease are prone to developing a complication of hyperhomocysteinemia, manifesting as an elevation of the homocysteine (Hcy) concentration in human plasma. However, Hcy as a protein-bound toxin is barely removed by conventional hemodialysis membranes. Here, we report a novel hemodialysis membrane by preparing a bioactive coating of pyridoxal 5'-phosphate (PLP) and adding biocompatible hyperbranched polyglycerol (HPG) brushes to achieve Hcy removal. The dip-applied PLP coating, a coenzyme with a role in Hcy metabolism, dramatically promoted a decrease in the Hcy concentration in human plasma. Moreover, the aldehyde group of PLP had an intrinsic chemical reactivity toward the terminal amino group to immobilize the HPG brushes on the hemodialysis membrane surface. The hierarchical PLP-HPG layer-functionalized membranes had a high efficacy for eliminating Hcy, with a concentration from the initial stage of 150 µmol/L reduced to a nearly normal level of 20 µmol/L in simulated dialysis. By analyzing the impact of HPG brushes with various chain lengths, we found that HPG brushes with a medium length enabled the PLP coating with the bioactive function of Hcy conversion to additionally protect Hcy-attacked target cells by providing excellent hydrophilicity and a dense enough chain volume overlap of the hyperbranched architecture. Simultaneously, the densely packed HPG brushes generated a maximal steric and hydration barrier that significantly improved biofouling resistance against blood proteins. The optimally functionalized membranes showed a clearance of 83.1% urea and 49.6% lysozyme and a rejection of 96.0% bovine serum albumin. The diversely functionalized PLP-HPG layers demonstrate a potential route for a more integrated hemodialysis membrane that can cope with the urgent issue of hyperhomocysteinemia in clinical hemodialysis therapy.

Construction and Application of PLP Self-sufficient Biocatalysis System for Threonine Aldolase.

A number of organic synthesis involve threonine aldolase (TA), a pyridoxal phosphate (PLP)-dependent enzyme. Although the addition of exogenous PLP is necessary for the reactions, it increases the cost and complicates the purification of the product. This work constructed a PLP self-sufficient biocatalysis system for TA, which included an improvement of the intracellular PLP level and co-immobilization of TA with PLP. Engineered strain BL-ST was constructed by introducing PLP synthase PdxS/T to Escherichia coli BL21(ED3). The intracellular PLP concentration of the strain increased approximately fivefold to 48.5 µmol/gDCW. l-TA, from Bacillus nealsonii (BnLTA), was co-expressed in the strain BL-ST with PdxS/T, resulting in the engineered strain BL-BnLTA-ST. Compared with the control strain BL-BnLTA (254.1 U/L), the enzyme activity of the strain BL-BnLTA-ST reached 1518.4 U/L without the addition of exogenous PLP. An efficient co-immobilization system was then designed. The epoxy resin LX-1000HFA wrapped by polyethyleneimine (PEI) acted as a carrier to immobilize the crude enzyme solution of the strain BL-BnLTA-ST mixed with an extra 100 µM of exogenous PLP, resulting in the catalyst HFAPEI-BnLTA-STPLP 100. HFAPEI-BnLTA-STPLP 100 exhibited a half-life of approximately 450 h, and the application of the catalyst in the continuous biosynthesis of 3-[4-(methylsulfonyl) phenyl] serine had more than 180 batch reactions (>60%conv) without the extra addition of exogenous PLP. The excellent compatibility and stability of the system were further confirmed by other TAs. This work introduced a PLP self-sufficient biocatalysis system that can reduce the cost of PLP and contribute to the industrial application of TA. In addition, the system may also be applied in other PLP-dependent enzymes.

An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme.

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.

Purification and partial characterization of N-hydroxy-l-phenylalanine decarboxylase/oxidase from Bacillus sp. strain OxB-1, an enzyme involved in aldoxime biosynthesis in the "aldoxime-nitrile pathway".

An enzyme that catalyzes the conversion of N-hydroxy-l-phenylalanine to phenylacetaldoxime was shown to be present in the Z-phenylacetaldoxime-degrading bacterium, Bacillus sp. strain OxB-1. The aldoxime-forming enzyme, which is induced by L-phenylalanine, was purified 8,050-fold to apparent homogeneity with a yield of 15.2%. The enzyme has a subunit M(r) of about 86,000. The enzyme converts N-hydroxy-L-phenylalanine (K(m) 0.99 mM) to only one geometrical isomer, namely Z-phenylacetaldoxime. Relatively large amounts of pyridoxal 5'-phosphate (PLP) are required to be present in the reaction mixture because PLP reacts non-enzymatically with the N-hydroxy amino acid substrate to form a nitrone. Several characteristics of the enzyme were compared with those of other PLP-dependent aromatic amino acid-converting enzymes described in the literature. The enzyme is tentatively named "N-hydroxy-L-phenylalanine decarboxylase/oxidase". Finally, the possible biosynthesis and metabolism of phenylacetaldoxime in Bacillus sp. strain OxB-1 is discussed.

Role of Pyridoxal 5'-Phosphate at the Titanium Implant Interface In Vivo: Increased Hemophilicity, Inactive Platelet Adhesion, and Osteointegration.

Titanium is the most biocompatible inorganic biomaterial with a long history of use in orthopedic and dental implants. However, promoting rapid and effective bone formation and integration onto etched, rough TiO2 surfaces has been a challenging topic. Here, 21 commercially available molecules are examined that met the following criteria: (1) contain phosphonic acid for stable immobilization onto TiO2 surfaces and (2) have a molecular weight less than 500 Da for negligible coating thickness. Of these molecules, the surface immobilization of pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 , dramatically increases the hemophilic property of the surface and accelerated osteointegration in vivo. Analysis shows that PLP promotes surface binding of serum albumin and other plasma proteins by Schiff-base formations via its aldehyde group, providing a platform suitable for osteoblast adhesion. PLP also retards blood coagulation more than the widely used citric acid at the TiO2 surface. As PLP is capable of maintaining an inactivated status of surface-adsorbed platelets, delayed coagulation at the implant-blood interface allows for sufficient supply of growth factors from blood plasma and migration of osteoblasts. The results suggest that PLP can be widely applicable as a biocompatible, effective coating compound to promote osteointegration of titanium-based implants.

The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights.

Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, ß = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II' at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Šresolution for forms I, II and II', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

Physico-chemical and pharmacological comparison of pyridoxylated hemoglobin bound to polyoxyethylene or polymerized by glutaraldehyde.

Two modified hemoglobin solutions were assessed using the same physico-chemical and pharmacological techniques. The first was prepared by covalent binding of monomethoxypolyoxyethylene (MPOE) 1.9 kDa to pyridoxylated hemoglobin (PLP-Hb). The resulting conjugate had a molecular size of 100 kDa (MPOE-PLP-Hb). The solution was cleared of non-fixed MPOE through ion exchange chromatography on Spherodex, thus bringing viscosity and oncotic pressure back to physiological values. The second was prepared by limited polymerization of pyridoxylated hemoglobin with glutaraldehyde (POLY-PLP-Hb). Tangential flow ultrafiltration achieved a satisfactory polymer/oligomer return. Quality controls showed no difference between the solutions. Total isovolemic exsanguinotransfusions in the rat did not help differentiate the two solutions. Hemorrhagic shock (80% of blood volume, rat) gave definitive survival for 8 of the 14 animals tested with MPOE-PLP-Hb (57%) but only 3 of the 8 animals tested with POLY-PLP-Hb (38%). None of the chemical approaches to reduce hemoglobin loss proved any more efficient than another, with the evaluation techniques employed.

Molecular characterization of novel pyridoxal-5'-phosphate-dependent enzymes from the human microbiome.

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.

Probing the role of Tyr 64 of Treponema denticola cystalysin by site-directed mutagenesis and kinetic studies.

Tyr 64, hydrogen-bonded to coenzyme phosphate in Treponema denticola cystalysin, was changed to alanine by site-directed mutagenesis. Spectroscopic and kinetic properties of the Tyr 64 mutant were investigated in an effort to explore the differences in coenzyme structure and kinetic mechanism relative to those of the wild-type enzyme. The wild type displays coenzyme absorbance bands at 418 and 320 nm, previously attributed to ketoenamine and substituted aldamine, respectively. The Tyr 64 mutant exhibits absorption maxima at 412 and 325 nm. However, the fluorescence characteristics of the latter band are consistent with its assignment to the enolimine form of the Schiff base. pK(spec) values of approximately 8.3 and approximately 6.5 were observed in a pH titration of the wild-type and mutant coenzyme absorbances, respectively. Thus, Tyr 64 is probably the residue involved in the nucleophilic attack on C4' of pyridoxal 5'-phosphate (PLP) in the internal aldimine. Although the Tyr 64 mutant exhibits a lower affinity for PLP and lower turnover numbers for alpha,beta-elimination and racemization than the wild type, the pH profiles for their Kd(PLP) and kinetic parameters are very similar. Rapid scanning stopped-flow and chemical quench experiments suggest that, in contrast to the wild type, for which the rate-determining step of alpha,beta-elimination of beta-chloro-L-alanine is the release of pyruvate, the rate-determining step for the mutant in the same reaction is the formation of alpha-aminoacrylate. Altogether, these results provide new insights into the catalytic mechanism of cystalysin and highlight the functional role of Tyr 64.

Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-A resolution.

The molecular structure of alanine racemase from Bacillus stearothermophilus was determined by X-ray crystallography to a resolution of 1.9 A. The alanine racemase monomer is composed of two domains, an eight-stranded alpha/beta barrel at the N-terminus, which includes residues 1-240, and a C-terminal domain essentially composed of beta-strand (residues 241-388). In the structure of the dimer the mouth of the alpha/beta barrel of one monomer faces the second domain of the other monomer. The pyridoxal 5'-phosphate (PLP) cofactor lies in and above the mouth of the alpha/beta barrel and is covalently linked via an aldimine linkage to Lys39, which is at the C-terminus of the first beta-strand of the alpha/beta barrel. This is the first example of a PLP cofactor binding in the active site of a alpha/beta barrel. A number of other residues are involved in maintaining the position of the PLP in the protein. Of these, Arg219 is the most interesting, as it forms a hydrogen bond with the pyridine nitrogen of the cofactor. This is the first known occurrence of such an interaction with PLP and is expected to influence the electron delocalization in the PLP-alanine intermediates. A second arginine residue, Arg136, donates a hydrogen bond to the phenolic oxygen of PLP and may be involved in the binding of substrate as well as stabilization of intermediates. Finally, Tyr265', from the second monomer, is postulated to be 2 proton donor to the carbanion intermediate.

A natural compound (reuterin) produced by Lactobacillus reuteri for hemoglobin polymerization as a blood substitute.

Stroma-free hemoglobin (Hb) has been modified by pyridoxylation and followed by polymerization with glutaraldehyde as a blood substitute. Nevertheless, the reaction rate of pyridoxylated Hb (PLP-Hb) with glutaraldehyde is too fast to control its molecular weight distribution. Additionally, it was reported that glutaraldehyde is cytotoxic even at low doses. To overcome these problems, another aldehyde, beta-hydroxypropionaldehyde (beta-HPA), was used in the study to polymerize hemoglobin (PLP-Hb). beta-HPA is a natural compound (reuterin) produced by Lactobacillus reuteri. It was found that the maximum degree of PLP-Hb polymerization by reuterin (RR-PLP-Hb) was approximately 40% if the formation of high molecular (> 500 kDa) polymers should be prevented. In contrast, at the same reaction condition, the glutaraldehyde-polymerized PLP-Hb solution became gel-like, due to overpolymerization. This indicated that the rate of PLP-Hb polymerization by reuterin was significantly slower than that by glutaraldehyde. With increasing the reaction temperature, PLP-Hb concentration, or reuterin-to-PLP-Hb molar ratio, the time to reach the maximum degree of PLP-Hb polymerization by reuterin became significantly shorter. Removal of unpolymerized PLP-Hb from the RR-PLP-Hb solution can be effectively achieved by a gel-filtration column. The P(50) value of the unmodified Hb solution was 14 torr, while that of the RR-PLP-Hb solution was 20 torr, an indication of lower oxygen affinity. Additionally, the oxygen-Hb dissociation curves for both test solutions had a sigmodial shape and a nearly 100% saturation at 100 torr. In the in vivo study, it was found that the animals treated with the RR-PLP-Hb solution all survived and remained healthy more than 3 months. In contrast, only one out of six rats survived for the control group treated with the unmodified Hb solution. Furthermore, it was found that the RR-PLP-Hb solution resulted in a significantly longer circulation time ( approximately 12 h) than the unmodified Hb solution ( approximately 1.5 h). These results suggest that the reuterin-polymerized PLP-Hb solution may be a new option in the development of blood substitutes.

Synthesis and characterization of artificial red cell (ARC).

An unsaturated lipid made from 2,4-octadecadienoic acid was used as a component of phospholipid vesicles. The vesicles (0.2 micron phi) was prepared by an extrusion method, and the unsaturated lipid was polymerized with gamma-ray. Vesicles constructed from the polylipid show enormous stability during long term storage in frozen state and can be handled easily. Negative effect could not be confirmed in vitro tests. This system satisfies all the physiological conditions, such as oxygen transport ability and solution properties as a blood substitute.

The development of hemoglobin solutions as red cell substitutes: hemoglobin solutions.

Although the efficacy of hemoglobin-based oxygen carriers was established more than 60 years ago, all prior clinical trials have demonstrated significant toxicity characterized by renal dysfunction, gastrointestinal distress, and systemic vasoconstriction. The mechanisms of these toxicities now appear to be understood. Tetrameric forms of the hemoglobin molecule extravasate from the circulation and interact with endothelial derived relaxing factor, leading to unopposed vasoconstriction. Although numerous efforts are underway to chemically modify the native tetramer, it is likely that all tetrameric forms of the hemoglobin molecule will continue to extravasate. We have focused on developing a polymerized form of hemoglobin that is virtually free of unreacted tetramer. The development and characterization of this polymerized pyridoxylated hemoglobin solution (Poly SFH-P) is described. Clinical trials have been completed successfully in volunteers, and are now underway to assess the safety and efficacy of Poly SFH-P as a clinically useful red cell substitute in the treatment of acute blood loss in the setting of trauma and surgery.