Vitamin B6: a long known compound of surprising complexity.
ABSTRACT In recent years vitamin B6 has become a focus of research describing the compound's critical function in cellular metabolism and stress response. For many years the sole function of vitamin B6 was considered to be that of an enzymatic cofactor. However, recently it became clear that it is also a potent antioxidant that effectively quenches reactive oxygen species and is thus of high importance for cellular well-being. In view of the recent findings, the current review takes a look back and summarizes the discovery of vitamin B6 and the elucidation of its structure and biosynthetic pathways. It provides a detailed overview on vitamin B6 both as a cofactor and a protective compound. Besides these general characteristics of the vitamin, the review also outlines the current literature on vitamin B6 derivatives and elaborates on recent findings that provide new insights into transport and catabolism of the compound and on its impact on human health.
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ABSTRACT: Lactobacillus bulgaricus is commonly used in dairy products as a starter culture. Its viability during freeze-drying is of commercial interest. Here a significant (p < 0.05) improvement in survival rate of L. bulgaricus ATCC 11842 was achieved during freeze-drying when it was prestressed with 2 % (w/v) NaCl for 2 h in the late growth phase. To understand the mechanism of this stress-related viability improvement in L. bulgaricus, protein synthesis was analyzed by 2D difference gel electrophoresis. Nine protein spots were significantly altered by NaCl and were subsequently identified by peptide mass fingerprinting. The functions of the proteins included stress-related protein synthesis, amino acid biosynthesis, nucleotide biosynthesis, sugar metabolism, transport systems, and vitamin biosynthesis. These findings provide a considerable background regarding the NaCl stress response of L. bulgaricus.Biotechnology Letters 07/2014; · 1.74 Impact Factor
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ABSTRACT: Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5'-phosphates. Pyridoxal 5'-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5'-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti, were assembled to two expression cassettes and introduced into the B. subtilis chromosome. In vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis.Metabolic Engineering 06/2014; · 8.26 Impact Factor
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ABSTRACT: Serine hydroxymethyltransferase isoforms (SHMT1 & SHMT2α), which serve as scaffold protein for the formation of a multi-enzyme complex and generate one-carbon unit for the de novo thymidylate biosynthesis pathway during DNA synthesis, are vitamin B6 (VB6)-dependent enzyme. Cancer cells with high proliferation intensity need increased SHMT activation which enforces the facilitated-diffusion of VB6 for the continuous functioning of thymidylate synthase cycle. Therefore, SHMT knockdown presents an alternative approach to prevent DNA synthesis in cancer cells; however, its potential to inhibit cancer growth remains unknown so far. Here we demonstrated that VB6 coupled to poly(ester amine) (VBPEA) enforces a high level of VTC (VB6-transporting membrane carriers)-mediated endocytosis of the complexed SHMT1 siRNA (siSHMT1) to interrupt the thymidylate biosynthesis pathway of cancer cells. The detrimental effect of SHMT1 knockdown on the disintegration of multi-enzyme complex resulted in cell cycle arrest and a decrease in cell's genomic DNA content, leading to enhanced apoptotic events in cancer cells. A reduction in tumor size was observed with constant SHMT1 suppression in xenograft mice. This study illustrates how silencing the SHMT1 expression inhibits cancer growth and the increased VB6 channeling for sustenance of cancer cells promotes VB6-coupled vector to elicit enhanced delivery of siSHMT1.Biomaterials 08/2014; · 8.31 Impact Factor
Molecules 2009, 14, 329-351; doi:10.3390/molecules14010329
Vitamin B6: A Long Known Compound of Surprising
Sutton Mooney 1, †, Jan-Erik Leuendorf 2, †, Christopher Hendrickson 1 and Hanjo Hellmann 1,*
1 School of Biological Sciences, Washington State University, Pullman, WA, USA; E-mail:
email@example.com (S. M.), firstname.lastname@example.org (C. H.)
2 Angewandte Genetik, Freie Universität Berlin, 14195 Berlin, Germany
E-mail: email@example.com (J-E. L.)
† These authors contributed equally to this work
* Author to whom correspondence should be addressed; E-mail: firstname.lastname@example.org.
Received: 17 December 2008; in revised form: 7 January 2009 / Accepted: 9 January 2009 /
Published: 12 January 2009
Abstract: In recent years vitamin B6 has become a focus of research describing the
compound’s critical function in cellular metabolism and stress response. For many years
the sole function of vitamin B6 was considered to be that of an enzymatic cofactor.
However, recently it became clear that it is also a potent antioxidant that effectively
quenches reactive oxygen species and is thus of high importance for cellular well-being. In
view of the recent findings, the current review takes a look back and summarizes the
discovery of vitamin B6 and the elucidation of its structure and biosynthetic pathways. It
provides a detailed overview on vitamin B6 both as a cofactor and a protective compound.
Besides these general characteristics of the vitamin, the review also outlines the current
literature on vitamin B6 derivatives and elaborates on recent findings that provide new
insights into transport and catabolism of the compound and on its impact on human health.
Keywords: Pyridoxal phosphate; Vitamin B6; Oxidative stress; Derivatives; Transporter.
Molecules 2009, 14
The Discovery of Vitamin B6
The formula of vitamin B6 (henceforth referred to as vitB6) was first published by Ohdake in 1932.
He worked on the isolation from rice-polishings of what he called “Oryzanin” (Vitamin B1) and found
vitB6 as a by-product [1, 2]. Ohdake described the formula, but he was not aware that he had found a
vitamin and did not recognize its physiological importance.
At this time several scientists worked on the characterization of the vitamin B family members [3-
10]. These scientists were searching for the so called “rat pellagra prevention factor” that could cure
acrodyna, a pellagra-like skin disorder in rats. They discovered that by adding a special yeast eluate to
the diet, acrodyna could be cured. Paul György, a Hungarian born scientist, first described vitB6 as the
active “rat pellagra prevention factor” in the yeast eluate . Several years later in 1938 five separate
groups of researchers, including György, isolated the crystalline vitB6 from yeast [4-8]. After
determination of the structure of vitB6 in 1939, György named the vitamin pyridoxine due to its
structural homology to pyridine . In the same year, Stanton A. Harris and Karl Folkers,
accomplished the synthesis of vitB6 .
In further studies it was shown that vitB6 could exist in other chemical forms that differ from
pyridoxine by a variable group present at the 4′ position . Pyridoxine (PN) carries a hydroxyl,
pyridoxal (PL) an aldehyde and pyridoxamine (PM) an amino group (Figure 1). While all three species
can be phosphorylated, it is pyridoxal 5’-phosphate (PLP) that is the biologically most active form and
used as cofactor for many important enzymatic reactions.
The Function of VitB6
The discovery and the first publications on vitB6 ascribed a growth function to the vitamers [3, 11].
However, further studies clarified that this relatively rough description understated the diversity of
crucial functions and importance for living organism that vitB6 has. VitB6, in the form of PLP, plays a
primary role acting as a cofactor for a large number of essential enzymes. These PLP-dependent
enzymes catalyze more than 140 distinct enzymatic reactions and belong to five (oxidoreductases EC
1, transferases EC 2, hydrolases EC 3, lyases EC 4, isomerases EC 5) of the six enzyme classes
defined by the Enzyme Nomenclature Committee of the International Union of Biochemistry and
Molecular Biology (http://www.chem.qmul.ac.uk/iubmb/enzyme). This underlines the wide variety of
chemical reactions that PLP-dependent enzymes promote in the organisms and shows again the
importance of vitB6. The following section will give an overview of the metabolic reactions in which
PLP-dependent enzymes are significantly involved.
Many of the PLP-dependent enzymes catalyze important steps in the amino acid metabolism, like
co-catalyzing transamination, racemization, decarboxylation, and α,β-elimination reactions [12, 13].
For example, transaminases mediate the conversion of α-ketoacids to amino acids and amino acid
racemases produce D-amino acids from L-amino acids .
Another site of action for the PLP-dependent enzymes is fatty acid metabolism. The enzyme δ-6-
desaturase (EC 188.8.131.52) catalyzes the synthesis of vital polyunsaturated fatty acids by the
desaturation of linolic acid and γ-linolenic acid, respectively [15, 16].
Molecules 2009, 14
Besides these roles, PLP also represents an important cofactor for the degradation of storage
carbohydrates, such as glycogen. The PLP-dependent glycogen phosphorylase (EC 184.108.40.206) mediates
the glycogen breakdown by the release of glucose from glycogen .
Furthermore, two PLP-dependent enzymes are involved in hemoglobin formation and chlorophyll
biosynthesis. In these reactions the rate-limiting step is the primary biosynthesis of δ-aminolevulinic
acid. In mammals and birds δ-aminolevulinic acid is synthesized by the action of δ-aminolevulinic
acid synthase (EC 220.127.116.11) and in plants and algae by the action of glutamate-1-semialdehyde 2,1-
aminomutase (EC 18.104.22.168) .
Additionally, in plants the biosynthesis of the phytohormone ethylene is controlled by the synthesis
of the precursor 1-aminocyclopropane-1-carboxylic acid from S-adenosylmethionine by PLP-
dependent 1-aminocyclopropane-1-carboxylate synthases (EC 22.214.171.124) .
Apart from its function as a cofactor for PLP-dependent enzymes, vitB6 is also thought to act
directly as a protective agent against reactive oxygen species, such as singlet oxygen which will be
discussed in a following section [20, 21].
While fungi, plants, archae, and most eubacteria are able to synthesize vitB6 (see next Section),
most animals, including humans, lack this ability and rely on the external supply of vitB6.
The Known Pathways of VitB6 Anabolism
Deoxyxylose 5’-phosphate-Dependent and –Independent De Novo Biosynthesis of VitB6
Two existing pathways are known for de novo vitB6 biosynthesis. First, the deoxyxylose 5’-
phosphate (DXP)-dependent pathway, which is present in eubacteria, such as Escherichia coli and
second the DXP-independent pathway which is described for some bacteria, archaea and eukarya.
The DXP-dependent pathway has been intensively studied in the gram-negative bacterium E. coli
. It was shown that in E. coli, vitB6 is synthesized by the action of PdxJ (EC 126.96.36.199) and PdxA
(EC 188.8.131.522) (Figure 1) [23-25]. These two vitB6 synthase proteins use 4-phospohydroxy-L-
threonine (4HPT) and DXP, which are precursors in isoprenoid and thiamine biosynthesis,
respectively, as substrates to form PNP [26-28]. PdxA catalyzes the oxidation of 4HPT to 3-amino-1-
hydroxyacetone 1-phosphate (AHAP), and PdxJ forms PNP with the intermediates AHAP and DXP
. PNP is then oxidized to PLP, the biocatalytically active form of vitB6, by PdxH via the salvage
pathway (Figure 1) . The vitB6 precursors 4HPT and DXP originate on the one hand from the
oxidation plus transamination of D-erythrose-4-phosphate, and on the other hand, by the synthesis
from pyruvate and D-glyceraldehyde-3-phosphate by DXP synthase (EC 184.108.40.206) .
Analysis of the crystal structure of the participating enzymes showed that PdxA and PdxJ act
separately. PdxA dimers create an interface onto which 4HPT binds . In contrast PdxJ forms
octamers as tetramers of PdxJ dimers . In every dimer interface a pocket is located where the
intermediates DXP and AHAP are converted to PNP.
The second known de novo vitB6 biosynthesis pathway is the DXP-independent pathway, which is
found in bacteria, archaea, and eukarya [13, 33, 34]. The occurrence of this pathway is demonstrated in
plants, fungi, Plasmodium falciparum, Thermotoga maritima as well as Bacillus subtilis and involves
two proteins, PDX1 and PDX2 (for pyridoxine biosynthesis protein; orthologs for B. subtilis YaaD and
YaaE, Geobacillus stearothermophilus PDXS and PDXT, Saccharomyces cerevisiae SNZ and SNO)
Molecules 2009, 14
[33-37]. These two synthase proteins directly synthesized PLP from ribose 5’-phosphate or ribulose 5’-
phosphate, in combination with either glyceraldehyde 3’-phosphate or dihydroxyacetone phosphate
and glutamine (Figure 1). Here PDX2 acts as a glutaminase, which deaminates glutamine to glutamate
in order to supply nitrogen for the PLP heterocycle, and then PDX1 arranges the final ring closure [33,
Figure 1. Schematic drawing of de novo and salvage pathways.
Crystallization studies in the organisms B. subtilis, G. stearothermophilus and P. falciparum
demonstrated that the PDX enzymes form a synthase complex with a cogwheel-like structure [37, 39,
40]. The core of the PLP synthase complex consists of 12 PDX1 enzymes that interact in two
hexameric layers joining face-to-face to form a dodecamer onto which 12 PDX2 monomers attach [37,
The Salvage Pathway
In addition to the direct synthesis of new PNP or PLP, the vitamers are interconvertible via the so
called salvage pathway. These conversions are accomplished by the action of either kinases or
oxidases [22, 41, 42]. The salvage pathway has been best analyzed in E. coli, where it has been shown
that two different kinases (EC 220.127.116.11) can phosphorylate PN, PL and PM to their respective 5’-
phosphates (Figure 1). The two kinases differ in their substrate specifities, with PdxY acting on PL,
whereas PdxK can utilize all three non-phosphorylated vitamers as substrates. Most eukaryotes contain
a single kinase, and crystal structure of the kinase from several different organisms has revealed a
dimer [43-46]. Each of the two monomers contains an active site that utilizes bound ATP and metal
ions, which are required for activity .
Contrary to the kinases, only one oxidase, PdxH (EC 18.104.22.168), is shown in E. coli to oxidize the
phosphorylated forms of PNP and PMP to PLP (Figure 1) [28, 48].
Molecules 2009, 14
In yeast a pyridoxine phosphate oxidase, PDX3 (EC 22.214.171.124), was identified and mutants in this
gene had increased oxidative stress sensitivities . Interestingly, complementation with a recently
identified oxidase in Arabidopsis, AtPPOX, rescued this yeast mutant which underscores the highly
conserved nature of the pathway . The crystal structure of pyridoxine oxidase has shown that this
protein also functions as a dimer, with the cofactor flavin mononucleotide, FMN, bound in the active
site of each monomer [51-53].
The Other End: VitB6 Catabolism
Besides the biosynthesis of vitB6, catabolism of the vitamin is also an important aspect for cellular
homeostasis of the compound. A critical step is represented by the dephosphorylation of
PLP/PMP/PNP because this step represents a major control for the pool of available active vitB6
cofactor. There are reports of unspecific dephosphorylation of PLP/PMP/PNP by alkaline phosphatase
(EC 126.96.36.199) and acid phosphatase (EC 188.8.131.52) [54-58]. However, additional phosphatases have been
annotated for various organisms that specifically target phosphorylated vitB6 as a substrate [59, 60].
Of these currently the one best characterized is human pyridoxal phosphatase (PLPP) (EC 3.1.3)
(Figure 2). The enzyme is a 64 kDa dimer with a requirement for Mg2+. It is expressed in various
tissues but predominantly in brain, liver and testis . PLPP has its highest affinity for PLP, followed
by PNP and then PMP. Inorganic phosphate has a strong inhibitory effect, but the enzyme can also be
weakly inhibited by PL [60, 61]. Although basic biochemical data are well established for human
PLPP control of its activity is still open.
Figure 2. Bacterial catabolic pathways of vitB6. Roman numbers in parenthesis indicate
enzymes: (I) pyridoxine 4-oxidase; (II) pyridoxal phosphatase; (III) pyridoxal 4-
dehydrogenase; (IV) 4-pyridoxollactonase; (V) pyridoxol oxidase. Arabic numbers indicate
compound: (1) PN; (2) PLP; (3) PL; (4) 4-pyridoxolactone; (5) 4-pyridoxic acid; (6)
succinic semialdehyde (modified from ).
Molecules 2009, 14
In the gram-negative bacteria Pseudomonas sp. and Mesorhizobium loti a detailed pathway has
been described for degradation of vitB6 and its phosphorylated derivatives (see Figure 2 for an
overview) [62, 63]. Here PL can either be converted in a two-step process from 4-pyridoxolactone to
4-pyridoxic acid (4-PA) via the subsequent activities of pyridoxal-4-dehydrogenase (EC 184.108.40.206) and
4-pyridoxolactonase (EC 220.127.116.11) or directly to 4-PA by pyridoxal oxidase (EC 18.104.22.168); 4-PA in turn
is then catabolized in five additional steps to succinic semialdehyde [62, 64]. It is noteworthy that 4-
PA is also found in rats and human which makes it likely that similar catabolic pathways exist in these
organisms [65, 66]. In addition, succinic semialdehyde is a common compound that can accumulate in
humans if, for example, degradation of γ-amino-butyric acid is impaired by succinic semialdehyde
dehydrogenase (EC 22.214.171.124) deficiency . However, it is basically unknown how the vitamin is
degraded in eukaryotes after pyridoxine phosphatase activity.
Allocation of VitB6 in Prokaryotes and Eukaryotes
A highly important but poorly understood field is the translocation of vitB6 in prokaryotes and
eukaryotes. Some vitB6 auxotrophic prokaryotes and single cell eukaryotes rely on the import of the
vitamin, while multicellular eukaryotes that cannot synthesize vitB6 need to translocate the vitamin to
their different organs.
Pioneering studies that demonstrated the existence of such vitB6 transporters came from Salmonella
typhimurium and S. cerevisiae [68, 69]. In higher eukaryotes studies on rat renal proximal tubular cells
demonstrated uptake of pyridoxine and N-(4'-pyridoxyl) amines . Here, the work by Zhang and
McCormick showed that the tested rat cells have an active Na+-dependent uptake system, which is able
to discriminate between the different offered substrates. Interestingly, both pyridoxine and its amine
derivatives were substrates of a pyridoxal kinase as they were phosphorylated upon entry into the
cytosol . Intestinal absorption in humans comes from two sources, dietary ingestion in the small
intestine and uptake of bacteria produced vitB6 in the large intestine [71, 72]. Experiments using
human intestinal epithelial Caco-2 cells and mammalian colonocytes demonstrated the involvement of
carrier-mediated systems for both with pH, temperature and pyridoxine levels affecting the rate of
transport. An intercellular protein kinase A (PKA)-mediated pathway appears to regulate transport in
the Caco-2 cells, whereas transport in colonocytes appears to be regulated by a Ca2+/CaM- mediated
pathway [71, 72]. Work in human placental tissue has also indicated the presence of a passive transport
system that transfers pyridoxine in and out of the cells but not against a concentration gradient .
Both active uptake and export of vitB6 have been demonstrated in yeast [69, 74, 75]. The fission yeast,
Schizosaccharomyces pombe, showed a preferential efflux of PN that was dependent on the internal
concentration of PN, and the rate was increased with the addition of Na+ . It was suggested that the
membrane potential could be affecting the PN efflux gate or carrier. Additionally, work in S. pombe
has linked the PL reductase encoded by prl1+ gene to the excretion of PN following its reduction from
PL as part of the maintenance of vitB6 levels in the cell . Overall these findings demonstrated the
presence of various mechanisms for transport of the vitB6. However the precise identity of the
transport proteins that facilitate such movements has remained open. Described for S. cerevisiae in
2003, Tpn1p was the first example of a eukaryotic vitB6 transporter . The protein belongs to the
family of purine-cytosine permeases and functions as a plasma membrane bound proton symporter for
vitB6 uptake. It has a high affinity for PN with a Km value of 0.55 μM but also transports PM and PL
Molecules 2009, 14
with lower affinities. A second unrelated transporter, Bsu1p was identified in S. pombe, a yeast strain
that does not contain a Tpn1p homlog . While Bsu1p has lower affinity for PN than Tpn1p, it also
operates as a proton symporter with similar optimal pH and substrate profiles. Likewise, expression of
both transporters increased when PN concentrations decreased [77, 78]. Very recently, a novel class of
vitamin transporters were identified in prokaryotes . They are composed of different modules that
have substrate specific components and an energy-coupling module and were named accordingly as
energy-coupling factor (ECF) transporters. The energy-coupling module allows docking of different
substrate specific components to facilitate selected transport of metabolites across membranes.
Interestingly, the authors also identified an ECF transporter with a high affinity for pyridoxine .
The findings of Tpn1p, Bsu1p and the ECF transporters demonstrate that different transport pathways
have evolved and it will be exciting to learn what kind of transport proteins are active in other
organisms. In addition it will be of importance to understand intracellular transport of vitB6 since
many PLP-dependent enzymes are present in mitochondria and chloroplast. However, currently it is
open how this is achieved in the cell since corresponding transport proteins remain to be identified.
The Role of VitB6 in Stress Response
Recent work has provided evidence of an expanded role of vitB6 in cells. As stated above, this
compound serves a role in enzymatic catalysis. However, vitB6 may play a crucial role in protecting
cells from oxidative stress because the vitamin has been shown to exhibit antioxidant activity that even
exceeds that of vitamins C and E [80-83]. Upon mutation of genes involved in the salvage and de novo
pathways of vitB6 synthesis, a range of phenotypes are seen in salt and reactive oxygen species (ROS)
sensitivity (Table 1). ROS sensitivity in context with vitB6 was originally characterized in the
phytopathogen Cercospora nicotianae. Here mutant strains were identified that were particularly
vulnerable to their own toxin cercosporin, a photosensitizer that produces singlet oxygen and
superoxide upon irradiation [35, 81]. Cloning of the mutant genes in C. nicotianae revealed that the
mutated fungi were affected in a PDX1 ortholog [35, 80, 81]. These findings were of importance since
so far vitB6 had not been mentioned in the context of singlet oxygen resistance . Subsequent
studies in other organisms also showed that vitB6 is crucial for oxidative stress tolerance and other
abiotic stressors. For example, loss of Arabidopsis thaliana PDX1.3 causes hypersensitivity towards
treatment with Rose Bengal, a ROS inducing chemical . Moreover Arabidopsis pdx1.3 mutants are
also hypersensitive towards salt and UV-B treatments [85, 86]. It is also interesting to note that
mutants in the salvage pathway show aberrant stress sensitivities, demonstrating that vitB6 metabolism
in general is critical for abiotic stress tolerance. For example, the Arabidopsis sos4 mutant that is
affected in pyridoxine kinase PDXK is highly sensitive to salt stress . However, unlike mutants
affected in PDX1 genes that have been demonstrated to contain reduced levels of vitB6, sos4 has
overall increased levels of the vitamin [86, 88-90]. Though the precise reason for increased vitB6
levels in sos4 remains to be answered, the example given demonstrates that vitB6 levels do not strictly
correlate with stress tolerance. Hence, one must question whether aberrant vitB6 levels are the primary
reason for the observed abiotic stress sensitivities.
It is noteworthy that PDX1 and PDXK gene expression is also regulated in response to abiotic
stress. For example, B. subtilis PDX1 has been demonstrated to be upregulated in the presence of the
singlet oxygen producer, methylene blue . In addition, PYRO A (a PDX1 homologue described in
Molecules 2009, 14
Aspergillus nidulans) and Arabidopsis PDX1.3 are upregulated after exposure to UV radiation [92,
93]. Also SNZ1, a S. cerevisiae PDX1 homologue, has been shown to be present in higher amounts
during the stationary growth phase in which cultures are more prone to oxidative stress [94, 95].
Findings in plants revealed that expression of Arabidopsis PDX1 genes is regulated by drought,
chilling, UV-B treatment, and ozone . Finally, SOS4 up-regulation has also been demonstrated as a
response to cold stress and abscisic acid (ABA) treatment .
Overall there appears to be a broad and beneficial effect of vitB6 on abiotic stress tolerance in the
cell, and stressors have been found to result in an increased amount of expression of genes involved in
vitB6 biosynthesis. Notably and as mentioned above there are some exceptions to the observation that
increased vitB6 availability is beneficial: Herrero and Daub observed negligible changes in vitB6
content in tobacco in response to salt stress, and Gonzalez and coworkers noted significantly higher
vitB6 levels in sos4 relative to wild type plants despite the mutant’s increased salt sensitivity .
Considering these findings, future work may aim to link additional signals to a response of altered
vitB6 production in cells. Additionally, characterization of the ROS quenching capacity and
regulation of vitB6 biosynthesis may help to solve the close relationships seen between the vitamin
and the described sources of stress.
Table 1. Examples of VitB6 De Novo and Salvage Pathway Mutants in Context with Stress.
Organism Mutant Pathway affected Phenotype
sor1/pdx1 De novo
sensitivity, loss of
snz1/pdx1 De novo
Reduced growth in
Reduced growth in
sno1/pdx2 De novo
Reduced aerial & root
growth, increased salt
Molecules 2009, 14
The Diversity of VitB6 Derivatives
As described in the preceding paragraphs, vitB6 is a well-investigated compound critical for many
cellular processes as either a central cofactor or as a potent antioxidant. However, it is noteworthy that
a variety of different PN, PM, and PL derivatives have been described, for which the precise function
is not understood (Table 2). These derivatives potentially have novel functions, and may be crucial to
fully appreciate the biological relevance of vitB6.
The best known of these derivatives is probably 4’-O-methylpyridoxine or ginkgotoxin from the
tree Ginkgo biloba [12, 97]. The compound has been found in different tissues with the highest
concentrations being present in seeds . Although it has been shown that the additional 4'-O-methyl
group most likely derives from methionine, and that both phosphorylated and non-phosphorylated
forms of pyridoxine are methylated, the biosynthetic pathway leading to 4’-O-methylpyridoxine is still
unresolved [98, 99]. Ingestion of the toxin can lead to Gin-nan-sitotoxism, epileptic convulsions, and
other neuronal disorders . As seeds from Ginkgo trees are a food source in China and Japan, and
extracts from leaves are used in pharmaceutical products, they represent a potential health risk. The
PLP-dependent enzyme glutamate decarboxylase (GAD), which is critical for synthesis of the
neurotransmitter GABA was discussed as a potential target of 4’-O-methylpyridoxine. However, there
is no clear evidence that ginkgotoxin significantly reduces GAD activity when present in
physiologically relevant concentrations . In contrast, recent work rather suggests that the toxin is
competing with PN/PM/PL for human pyridoxine kinase . This in turn might reduce the pool of
available PLP and PMP in the brain and negatively affect GAD activity and GABA biosynthesis .
Ginkgotoxin was also found in the African tree Albizia tanganyicensis [98-100] demonstrating that
the biosynthetic pathway leading to the formation of 4’-O-methylpyridoxine is not unique to Ginkgo.
Albizia tanganyicensis and its close relative Albizia julibrissin also synthesize other more complex
vitB6 derivatives (see Table 2) [100, 102]. Unfortunately, neither for ginkgotoxin nor for the other
Albizia derivatives could we find a biological function explaining why these compounds are
synthesized. A likely possibility is that they serve as protecting compounds against pathogens due to
their toxicity. This poses the attractive question as to what kind of mechanisms these plants employ to
protect their own metabolism against toxic vitB6 derivatives. For example, do they utilize specific
compartments or organelles for storage of their toxic compounds?
Another aspect that vitB6 derivatives have been brought in context with is the formation of
advanced glycation and lipoxygenation end-products (AGE and ALE, respectively). AGE and ALE
formation can occur in cells when reduced sugars (e.g. glucose, fructose) or polyunsaturated fatty acids
are abundant. In such a situation they can cross-react preferentially with lysine residues of proteins
[103, 104]. Accumulation of AGE and ALE is also caused by oxidative stress or overload of pathways
active in detoxification . Such end-products are often detrimental for protein function and,
especially in older tissues, might lead to severe damage. Hence, patients with diabetes or
atherosclerosis that have increased contents of blood sugar or blood lipids, respectively, suffer from
accumulation of AGEs and ALEs. Here, pyridoxamine is discussed to serve as a protecting compound
by bonding with fatty acids (see Table 2) and thereby effectively competing with proteins for ALE
formation [104, 105]. The vitamin is also discussed to serve as a protecting compound for AGE
formation which is of special interest to patients suffering from diabetes [106-108].
Molecules 2009, 14
A significant proportion of vitB6 (ranging from 5-80% of the total vitB6 content) in many fruits and
vegetables is glycosylated [109, 110]. Glycosylated vitB6 appears to be abundant in plants and has
been detected in soybean, rice, and Ginkgo [111-113]. Furthermore, in fungi β-fructosyl and β-
galactosyl compounds of pyridoxine have been found [114, 115]. It is likely that these derivatives of
vitB6 are not substrates of a pyridoxine kinase and thus are not accessible for metabolic utilization.
Consequently, specific β-glucosidases have been described in plants and human capable of removing
the sugar moiety, making the vitamin again accessible for salvage pathway enzymes [111, 116-118].
Although no precise explanation for the high amount of glycosylated vitB6 is provided in literature, a
possibility can be seen in context with AGE accumulation. Here, vitB6 might serve as a protecting
compound to prevent reaction of sugar with lysine residues of proteins. Alternatively, glycosylated
forms of vitB6 might serve as storage compounds of the vitamin and even carbohydrates that can be
mobilized upon demand. Overall the existence of such a diverse variety of vitB6 derivatives indicates
that the vitamin is involved in or employed for many other currently unknown processes.
Table 2. Examples of VitB6 Vitamers and Their Derivatives
Derivative Structure Function Organism
R1= -HC-O (PL)
R2= -CH2OH (PN)
Molecules 2009, 14
Table 2. Cont.
VitB6 Supplements and Human Health: Is it too much or not enough of a good thing?
The importance of vitB6 in human health is reflected in how actively it is studied in relationship to
a wide range of disease prevention and control. The current Recommended Dietary Allowance (RDS)
from the American National Institute of Health (NIH) for vitB6 is 2 milligrams per day with an
upward tolerance of 100 mg per day for adults. High doses can lead to peripheral sensory neuropathy
and nerve degeneration [119, 120]. These problems are generally reversible when supplementation is
stopped. Additionally some studies have suggested that increased levels of the B6 vitamers and some
derivatives can generate toxic photoproducts as a result of UV irradiation [121-123].
Most problems are stemming from deficiencies of the vitamin and there are several areas of interest
actively being pursued. Numerous clinical trials have been conducted to observe the broader impact of
vitB6 on human health problems such as cancer prevention and recovery and the amelioration of
neurological diseases. Also under investigation are the benefits of increased vitB6 through its role as a
cofactor in upstream processes that lead to problems involving heart disease, osteoporosis and
diabetes. Several of these topics are addressed in the following section.
Studies of vitB6 supplements in cancer prevention have yielded mixed results ranging from no
significant benefits seen in breast cancer to indications of protection against colorectal cancer . A
recent study on hairless mice given PN supplements was performed to see if increased vitB6 protected
Molecules 2009, 14
against UV-B induced skin tumors . It was reasoned that vitB6 would help quench reactive
oxygen species (ROS), which are associated with the development of cancer. Interestingly though,
while higher serum levels of PLP correlated with a higher dosage of dietary PN, neither the amount of
PLP nor oxidative stress markers in skin differed in relationship to the dosages. Additionally an
increase in tumor induction was seen in the mice given the higher doses of PN, correlating with
previous work [122, 123]. Interestingly decreased levels of vitB6 accompanied by increased levels of
oxidative stress were detected in red blood cells of non-small cell lung cancer patients, also
highlighting the vitamin’s potential benefits as an antioxidant .
Phosphorylated vitB6 is needed as a cofactor for neurotransmitter synthesis. However, studies on
elderly people have shown that a walking program has more benefits on cognitive improvement and
increased folate may help prevent Alzheimer’s diseases rather than vitB6 supplements [126-129].
While an association of low PLP and high symptoms of depression have been reported,
supplementation with vitB6 has not been shown to conclusively improve depression in older men [130,
131]. In schizophrenic patients with tardive dyskinsia, plasma levels of PLP were significantly lower
and treatment with vitB6 supplements reduced the symptoms of this disease along with another
schizophrenic associated disease, akathisia [132-134]. VitB6 is also studied as a potentially important
candidate to improve behavioural disorders of autistic children, although the precise impact of the
vitamin remains to be shown. [135-136].
VitB6 is involved in maintenance of normal homocysteine levels, and lower levels of homocysteine
are associated with lower rates of coronary heart disease and stroke . However studies are
conflicting as to whether giving supplements to lower the homocysteine levels improves protection
against these diseases [137-139]. High homocysteine levels have also been linked to osteoperosis and
bone fragility fractures. In vitro tissue experiments demonstrated that either decreasing vitB6 or
increasing homocysteine levels stimulated osteoclast activity, which leads to bone resorption .
Blood vitB6 levels are significantly decreased in diabetics. As vitB6 is a cofactor in tryptophan
catabolism, disruption of this pathway leads to increased levels of kynurenine metabolites which
inhibit insulin secretion and lower glucose tolerance . Studies on different B6 vitamers have
shown that supplements can help with problems related to glucose tolerance . Complications
from other diabetes-associated diseases are also common, and interestingly high doses of vitB6
normalized endothelial dysfunction, a precursor to vascular disease, in children with type 1 diabetes
Outlook and Perspectives
Though in the last years various aspects in vitB6 biosynthesis and the impact and benefits of the
vitamin for general metabolism have been explained, many questions remain unanswered. Five of
these are covered in the following section to provide an outlook on future opportunities related to this
important research field.
Do regulatory mechanisms exist that control PLP de novo biosynthesis? Although the two biosynthetic
pathways – DXP-dependent and –independent – have been resolved, regulatory mechanisms on the
transcriptional and posttranscriptional level to control PLP biosynthesis remain to be found. Although
transcriptional regulation of PDX1 genes has been described for various organisms after stress
Molecules 2009, 14
treatments, it remains open whether this in turn affects vitB6 levels [13, 144]. Considering the central
and indispensable role of PLP in metabolism one would expect that such regulatory switches exist.
These need to be connected with general metabolism, first because vitB6 exhibits such a central role as
a cofactor and second because the de novo biosynthetic machinery competes for the precursors
required for PLP synthesis with other pathways.
How are the salvage pathway and PLP phosphatase activities regulated? As for the de novo pathways,
it has not been explained how salvage pathway enzymes and PLP phosphatases are regulated. This is
surprising as pyridoxine kinase and PNP/PMP oxidase are crucial players in controlling vitB6
homeostasis and the availability of the active cofactor which might even occur in concert with PLP
phosphatases. However, only a few studies reveal insights on factors like ions, ATP, or end-products
that directly affect activities of these enzymes [61, 145-149], while the interplay between the different
proteins and the de novo pathway has not been addressed. Again, one would suggest that some higher
degree of active control is present that regulates the proteins of the salvage pathway and PLP
phosphatases upon demand.
What are the mechanisms of vitamin B6 translocation? For many organisms it is open how vitB6 is
translocated within the different organs and tissues. Although the non-phosphorylated forms can to
some extent go passively through membranes, this diffusion is most likely insufficient for long
distance allocation or rapid uptake of the vitamin when needed . Currently only a few examples
are given for vitB6 transporters in yeast and prokaryotes, making this an important question in other
organisms like animals or plants [77, 79].
How does vitamin B6 metabolism positively affect stress tolerance? For many organisms it has been
shown that mutants affected in either the salvage or the de novo pathway are hypersensitive towards
abiotic stress conditions. However, this hypersensitivity does not always correlate with the vitB6
content in the cell [86, 90]. Hence it is currently unclear and necessary to ask whether the amount of
vitB6 is the critical factor to protect against abiotic stress, whether it is the vitB6 homeostasis that is
important, or whether it is the proteins that participate in the different pathways that have additional
functions connected to stress alleviation.
Are there more de novo pathways present in yet unexplored organisms and how are vitB6 derivatives
formed? Currently it appears to be that there are just the two described de novo pathways, and no
evidence for additional biosynthetic pathways is at hand. Though one cannot exclude the possibility
for a third pathway, it is more likely that only the two described DXP-dependent and DXP-
independent pathways exist. In contrast the variety of existing vitB6 derivatives indicates an extensive
metabolic ability of organisms to modify vitB6. It will be interesting in the future to have more
knowledge generated on the enzymes that modify vitB6, about the biological purposes of these
compounds, and how these organisms protect themselves against potentially toxic derivatives.
Understanding these points may provide better approaches to utilizing the pharmaceutical potentials of
vitB6 and its derivatives for human health.
Molecules 2009, 14
We would like to thank our Russian collaborator Dr. Cleatus for critical reading. We also would
like to thank the DFG (DFG grant HE3224/7-1 to HH) for supporting this work.
References and Notes
1. Ohdake, S. Isolation of "Oryzanin" (Antineuritic Vitamin) from Rice-polishings. Bull. Agri.
Chem. Soc. Japan. 1932, 8, 11-46.
Wiardy, P.W. Crystalline Vitamin B6 (Adermin). Nature 1938, 142, 1158-1158.
György, P. Vitamin B2 and the Pellagra-like Dermatitis in Rats. Nature 1934, 133, 498-499.
György, P. Crystalline vitamin B6. J. Am. Chem. Soc. 1938, 60, 983-984.
Ichiba, A.; Michi, K. Cristalline vitamin B6. Sci. Pap. Inst. Phys. Chem. Res. (Tokyo) 1938, 34,
Keresztesy, J.C.; Stevens, J.R. Vitamin B-6. Proc. Soc. Exp. Biol. Med. 1938, 38, 64-65.
Kuhn, R.; Wendt, G. Über das antidermatitische Vitamin der Hefe. Ber. Chem. Ges. 1938, 71,
Lepkovsky. Crystalline factor I. Science 1938, 87, 169-170.
György, P.; Eckardt, R.E. Vitamin B6 and Skin Lesions in Rats. Nature 1939, 144, 512-512.
10. Harris, S.A.; Folkers, K. Synthetic vitamin B6. Science 1939, 89, 347.
11. Snell, E.E.; Guirard, B.M; Williams, R.J. Occurrence in natural products of a physiologically
active metabolite of pyridoxine. J. Biol. Chem. 1942, 143, 519-530.
12. Drewke, C.; Leistner, E. Biosynthesis of vitamin B6 and structurally related derivatives. Vitam.
Horm. 2001, 61, 121-155.
13. Mittenhuber, G. Phylogenetic analyses and comparative genomics of vitamin B6 (pyridoxine) and
pyridoxal phosphate biosynthesis pathways. J. Mol. Microbiol. Biotechnol. 2001, 3, 1-20.
14. Grogan, D.W. Temperature-sensitive murein synthesis in an Escherichia coli pdx mutant and the
role of alanine racemase. Arc. Microbiol. 1988, 150, 363-367.
15. Nakamura, M.T.; Nara, T.Y. Structure, function, and dietary regulation of delta-6, delta-5, and
delta-9 desaturases. Annu. Rev. Nutr. 2004, 24, 345-376.
16. Horrobin, D.F. Fatty acid metabolism in health and disease: the role of delta-6-desaturase. Am. J.
Clin. Nutr. 1993, 57, 732S-736S; discussion 736S-737S.
17. Helmreich, E.J. How pyridoxal 5'-phosphate could function in glycogen phosphorylase catalysis.
Biofactors 1992, 3, 159-172.
18. Cheltsov, A.V.; Guida, W.C.; Ferreira, G.C. Circular permutation of 5-aminolevulinate synthase:
effect on folding, conformational stability, and structure. J. Biol. Chem. 2003, 278, 27945-27955.
19. Rottmann, W.H.; Peter, G.F.; Oeller, P.W.; Keller, J.A., Shen, N.F.; Nagy, B.P.; Taylor, L.P.;
Campbell, A.D.; Theologis, A. 1-aminocyclopropane-1-carboxylate synthase in tomato is encoded
by a multigene family whose transcription is induced during fruit and floral senescence. J. Mol.
Biol. 1991, 222, 937-961.
20. Bilski, P.; Li, M.Y.; Ehrenshaft, M.; Daub, M.E.; Chignell, C.F. Vitamin B6 (Pyridoxine) and Its
Derivatives Are Efficient Singlet Oxygen Quenchers and Potential Fungal Antioxidants.
Photochem. Photobiol. 2000, 71, 129-134.
Molecules 2009, 14
21. Chen, H.; Xiong, L. Pyridoxine is required for post-embryonic root development and tolerance to
osmotic and oxidative stresses. Plant J. 2005, 44, 396-408.
22. Hill, R.E.; Himmeldirk, K.; Kennedy, I.A.; Pauloski, R.M.; Sayer, B.G.; Wolf, E.; Spenser, I.D.
The biogenetic anatomy of vitamin B6. A 13C NMR investigation of the biosynthesis of
pyridoxol in Escherichia coli. J. Biol. Chem. 1996, 271, 30426-30435.
23. Arenz, A.; Klein, M.; Fiehe, K.; Gross, J.; Drewke, C.; Hemscheidt, T.; Leistner, E Occurrence of
neurotoxic 4'-O-methylpyridoxine in Ginkgo biloba leaves, Ginkgo medications and Japanese
Ginkgo food. Planta Med. 1996, 62, 548-551.
24. Drewke, C.; Notheis, C.; Hansen, U.; Leistner, E.; Hemscheidt, T.; Hill, R.E.; Spenser, I.D.
Growth response to 4-hydroxy-L-threonine of Escherichia coli mutants blocked in vitamin B6
biosynthesis. FEBS Lett. 1993, 318, 125-128.
25. Laber, B.; Maurer, W.; Scharf, S.; Stepusin, K.; Schmidt, F.S. Vitamin B6 biosynthesis: formation
of pyridoxine 5'-phosphate from 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose-5-
phosphate by PdxA and PdxJ protein. FEBS Lett. 1999, 449, 45-48.
26. Cane, D.E.; Hsiung, Y.; Cornish, J.A.; Robinson, J.K.; Spenser, I.D. Biosynthesis of Vitamin B6:
The Oxidation of 4-(Phosphohydroxy)-l-threonine by PdxA. J. Am. Chem. Soc. 1998, 120, 1936-
27. Lam, H.M.; Winkler, M.E. Characterization of the complex pdxH-tyrS operon of Escherichia coli
K-12 and pleiotropic phenotypes caused by pdxH insertion mutations. J. Bacteriol. 1992, 174,
28. Zhao, G.; Winkler, M.E. 4-Phospho-hydroxy-L-threonine is an obligatory intermediate in
pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12. FEMS Microbiol. Lett.
1996, 135, 275-280.
29. Sivaraman, J.; Li, Y.; Banks, J.; Cane, D.E.; Matte, A.; Cygler, M. Crystal structure of
Escherichia coli PdxA, an enzyme involved in the pyridoxal phosphate biosynthesis pathway. J.
Biol. Chem. 2003, 278, 43682-43690.
30. Notheis, C.; Drewke, C.; Leistner, E. Purification and characterization of the pyridoxol-5'-
phosphate: oxygen oxidoreductase (deaminating) from Escherichia coli. Biochim. Biophys. Acta
1995, 1247, 265-271.
31. Sprenger, G.A.; Schorken, U.; Wiegert, T.; Grolle, S.; de Graaf, A.A.; Taylor, S.V.; Begley, T.P.;
Bringer-Meyer, S.; Sahm, H. Identification of a thiamin-dependent synthase in Escherichia coli
required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids,
thiamin, and pyridoxol. Proc. Natl. Acad. Sci. USA 1997, 94, 12857-12862.
32. Franco, M.G.; Laber, B.; Huber, R.; Clausen, T. Structural basis for the function of pyridoxine 5'-
phosphate synthase. Structure 2001, 9, 245-253.
33. Burns, K.E.; Xiang, Y.; Kinsland, C.L.; McLafferty, F.W.; Begley, T.P. Reconstitution and
biochemical characterization of a new pyridoxal-5'-phosphate biosynthetic pathway. J. Am. Chem.
Soc. 2005, 127, 3682-3683.
34. Tambasco-Studart, M.; Titiz, O.; Raschle, T.; Forster, G.; Amrhein, N.; Fitzpatrick, T.B. Vitamin
B6 biosynthesis in higher plants. Proc. Natl. Acad. Sci. USA 2005, 102, 13687-13692.