Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.     

Control of vitamin B 6 biosynthesis in Escherichia coli

Pyridoxineless mutants of Escherichia coli B which specifically require pyridoxal or pyridoxamine for growth can be divided into classes according to their growth responses in enriched media. Members of the slowest growing class synthesize vitamin B(6) at the fastest rates when starved for pyridoxal in glycerol minimal medium. After 80 min of synthesis at 4 x 10(-10) moles of vitamin B(6) per mg of cells per hr, the rate increases four- to fivefold and continues at the new rate for several hours. The shift to the new rate is prevented by chloramphenicol, thus suggesting that a derepression mechanism exists to control vitamin B(6) synthesis in addition to the previously discovered feedback control.     

Identification of pyridoxine 3-sulfate, pyridoxal 3-sulfate, and N-methylpyridoxine as major urinary metabolites of vitamin B6 in domestic cats

Preliminary studies of vitamin B6 metabolism in three adult domestic cats detected very little pyridoxic acid in the urine. At oral doses of 49 to 490 mumol of [14C]pyridoxine hydrochloride, 50% of the excreted dose occurred as pyridoxine 3-sulfate and 25% as N-methylpyridoxine. The identity of these two metabolites was confirmed by isolation from urine and comparison with known compounds. A third compound was identified as pyridoxal 3-sulfate on the basis of chromatographic behavior and fluorescent properties before and after hydrolysis. At pyridoxine intakes of 0.97 mumol/day, the concentration of pyridoxal 3-sulfate in the urine sometimes exceeded the concentration of pyridoxine 3-sulfate. Pyridoxic acid remained a minor urinary metabolite at pyridoxine intakes ranging from 0.97 to 490 mumol/day. Although sulfation of phenol groups and methylation of the ring nitrogen are well-known detoxication reactions, this appears to be the first time such reactions have been observed in normal metabolism of vitamin B6. These observations provide further evidence of the diversity of vitamin B6 metabolism between species. While such diversity complicates the extrapolation of data from animal studies to humans, it does provide a variety of models for examining the influences of various factors on vitamin B6 metabolism.     

Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

Vitamin B6 metabolism in microbes and approaches for fermentative production

Vitamin B6 is a designation for the six vitamers pyridoxal, pyridoxine, pyridoxamine, pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate, and pyridoxamine. PLP, being the most important B6 vitamer, serves as a cofactor for many proteins and enzymes. In contrast to other organisms, animals and humans have to ingest vitamin B6 with their food. Several disorders are associated with vitamin B6 deficiency. Moreover, pharmaceuticals interfere with metabolism of the cofactor, which also results in vitamin B6 deficiency. Therefore, vitamin B6 is a valuable compound for the pharmaceutical and the food industry. Although vitamin B6 is currently chemically synthesized, there is considerable interest on the industrial side to shift from chemical processes to sustainable fermentation technologies. Here, we review recent findings regarding biosynthesis and homeostasis of vitamin B6 and describe the approaches that have been made in the past to develop microbial production processes. Moreover, we will describe novel routes for vitamin B6 biosynthesis and discuss their potential for engineering bacteria that overproduce the commercially valuable substance. We also highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies to circumvent these limitations.     

Tpn1p,the plasma membrane vitamin B6 transporter of Saccharomyces cerevisiae

Pyridoxine (PN) is a metabolic precursor of pyridoxal phosphate that functions as a cofactor of many enzymes in amino acid metabolism. PN, pyridoxal, and pyridoxamine are collectively referred to as vitamin B6, and mammalian organisms depend on its uptake from the diet. In addition to the ability to use extracellular vitamin B6, most unicellular organisms are also capable of synthesizing PN to generate pyridoxal phosphate. Here, we report the isolation of Saccharomyces cerevisiae mutants that have lost the ability to transport PN across the plasma membrane. We used these mutants to isolate TPN1, the first known gene encoding a transport protein for vitamin B6. Tpn1p is a member of the purine-cytosine permease family within the major facilitator superfamily. The protein functions as a proton symporter, localizes to the plasma membrane, and has high affinity for PN. TPN1 mutants lost the ability to utilize extracellular PN, pyridoxal, and pyridoxamine, showing that there is no other transporter for vitamin B6 encoded in the genome. Amino acid substitutions that led to a loss of Tpn1p function localized to transmembrane domain 4 within the 12-transmembrane domain protein. Moreover, expression of TPN1 was regulated and increased with decreasing concentrations of vitamin B6 in the medium. We also provide evidence that of the highly conserved SNZ and SNO genes in S. cerevisiae, only the protein encoded by SNZ1 is required for vitamin B6 biosynthesis.     

Disruption of the plr1+ gene encoding pyridoxal reductase of Schizosaccharomyces pombe

Pyridoxal (PL) reductase encoded by the plr1(+) gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B(6), or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1 Delta) strain was constructed and its phenotype was examined. The plr1 Delta cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1 Delta strain became flocculent at the late stationary phase for an unknown reason. The plr1 Delta cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1(+) gene, which maintained the flow of "PL --> PN --> PNP --> PLP" in the salvage synthesis of PLP. The total vitamin B(6) and pyridoxamine 5'-phosphate contents in the plr1 Delta cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B(6) compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1 Delta cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1(+ )gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis.     

Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay

Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma.     

Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry

Methods have been developed for the simultaneous quantitative analysis of vitamin B6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry (GC/MS). The three resulting vitamers were analyzed by electron ionization GC/MS using a silicone capillary column. The methods have been applied to analysis of vitamin B6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.     

Identification of the Forms of Vitamin B6 Present in the Culture Media of “Vitamin B6 Control” Mutants

An Escherichia coli mutant resistant to isoniazid (WG497) contained 0.6 mumole of extracellular pyridoxamine and pyridoxamine phosphate in the early stationary phase. A suppressed lysine mutant (AT1024) contained 1.4 mumoles of pyridoxal phosphate under the same conditions. The internal concentration of vitamin B(6) was one-half of normal for AT1024 and increased fivefold for WG497.     

New synthesis of (+/-)-alpha-CMBHC and its confirmation as a metabolite of alpha-tocopherol (vitamin E)

There is currently interest in the metabolism of the various compounds which make up the vitamin E family, especially with regards to the possible use of vitamin E metabolites as markers of oxidative stress and adequate vitamin E supply. A number of vitamin E metabolites have been described to date and we have recently developed a method to extract and quantitate a range of vitamin E metabolites in human urine. During the development of this method a new metabolite of alpha-tocopherol was identified, which we tentatively characterised as 5-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-2-methyl-pentanoic acid (alpha-CMBHC).(1) Here we describe the synthesis of alpha-CMBHC as a standard and confirm that it is a metabolite of alpha-tocopherol.     

Isolation and identification of 4,25-dihydroxyvitamin D2: a novel A-ring hydroxylated metabolite of vitamin D2

Vitamin D2 is less toxic in rats when compared to vitamin D3. Our laboratory has been involved in research projects which were directed towards identifying the possible mechanisms responsible for the toxicity differences between vitamins D2 and D3 in rats. The present research project was designed to isolate and identify new metabolites of vitamin D2 from serum of rats which were fed toxic doses of vitamin D2. Hypervitaminosis D2 was induced in 30 rats by feeding each rat with 1000 nmol of vitamin D2/day x 14 days. The rats were sacrificed on the 15th day and obtained 180 ml of serum. The lipid extract of the serum was directly analyzed by a straight phase HPLC system. The various vitamin D2 metabolites were monitored by their ultraviolet (UV) absorbance at 254 nm. One of the UV absorbing peaks did not comigrate with any of the known vitamin D2 metabolites. This unknown metabolite peak was further purified by HPLC and was then subjected to UV absorption spectrophotometry and mass spectrometry. The structure assignment of the new metabolite was established to be 4,25-dihydroxyvitamin D2 [4,25(OH)2D2] by the techniques of UV absorption spectrophotometry and mass spectrometry and by the new metabolite's susceptibility to sodium metaperiodate oxidation. At present the biological activity of this unique 'A-ring' hydroxylated vitamin D2 metabolite is not known. As this new metabolite is isolated from the serum of rats intoxicated with vitamin D2, we speculate that 4,25(OH)2D2 may be playing an important role in the deactivation of vitamin D2.     

Vitamin B6-mediated suppression of colon tumorigenesis,cell proliferation,and angiogenesis (review)

This review describes current research on the preventive effect of dietary vitamin B(6) against colon tumorigenesis and its possible mechanisms. Studies in cell culture have demonstrated that high levels of vitamin B(6) suppress growth of some cancer cells. From these studies it has been considered that supraphysiological doses of vitamin B(6) suppress tumor growth and metastasis. However, recent rodent study has indicated that azoxymethane-induced colon tumorigenesis in mice is suppressed by moderate doses of dietary vitamin B(6.) Epidemiological studies also support an inverse relationship between vitamin B(6) intake and colon cancer risk. Potential mechanisms underlying the preventive effect of dietary vitamin B(6) have been suggested to include the suppression of cell proliferation, oxidative stress, nitric oxide (NO) synthesis, and angiogenesis.     

Transport and metabolism of vitamin B6 in lactic acid bacteria.

Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats.

The metabolism of [6-3H]pyridoxine - HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 muCi of [6-3H] pyridoxine - HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H+) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6 deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a platuau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continuous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.     

Isolation and identification of the glucuronide of 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid from rabbit urine

The metabolic profile of 3H-1,2-dihydro-2-(4-methylphenylamino)methyl-1-pyrrolizinone (SFZ-47), a putative non-steroidal anti-inflammatory pro-drug, has been studied in rabbit urine. Semi-preparative reversed-phase HPLC of 24 h urine from two rabbits given single oral doses of SFZ-47 (200 mg) allowed the separation of SFZ-47 together with the oxidative metabolite 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid (SFZ-47-COOH) and its glucuronide conjugate. The glucuronide was characterized by ESI-MS(n) and (1)H NMR and shown to be the 1-O-acyl beta-D-glucuronide conjugate of SFZ-47-COOH. The method gave excellent resolution of the glucuronide from endogenous constituents in urine and may be suitable for the preparation of glucuronide metabolites of other drugs.     

Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.     

Effect of Yeast Extract and Vitamin B(12) on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B(12), biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B(12) alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Photo-induced processes in vitamin B6 compounds

In this work, we applied multi-wavelength stopped-flow spectroscopy (MSFS) to study the chemical equilibria between tautomeric or hydrated forms of various vitamin B6 compounds and the Schiff base formed by epsilon-aminocaproic acid (= 6-aminohexanoic acid) with pyridoxal 5'-phosphate at 25 degrees and variable pH. Since some of these compounds are photosensitive, we analyzed the possible occurrence of any secondary photo-induced processes under the conditions of irradiation in the MSFS equipment (continuous irradiation with light from a 75-W Xe lamp spanning the wavelength range of 200-700 nm). To determine the tautomeric composition of these compounds, the electronic absorption spectra were analyzed by means of log-normal curves. Continuous irradiation of pyridoxamine and pyridoxal 5'-phosphate over the wavelength range of 200-700 nm displaces the chemical equilibrium between the tautomeric or hydrated forms of these compounds. However, the Schiff base of epsilon-aminocaproic acid with pyridoxal 5'-phosphate is insensitive to the radiation used. The photo-induced processes detected in pyridoxamine and pyridoxal 5'-phosphate should be taken into account in examining vitamers by MSFS. In fact, these additional processes should be considered in studying the mechanism of action of vitamin B6-dependent enzymes by the MSFS technique, whenever some free vitamer may be present in solution.