Czech J. Food Sci.

Vol. 25, No. 3: 101–118

Biosynthesis of Food Constituents: Vitamins.

2. Water-Soluble Vitamins: Part 2 – a Review

Jan Velíšek and Karel Cejpek

Department of Food Chemistry and Analysis, Faculty of Food

and Biochemical Technology, Institute of Chemical Technology in Prague,
Prague, Czech Republic

Abstract

Velíšek J., Cejpek K. (2007): Biosynthesis of food constituents: Vitamins. 2. Water-soluble vitamins:
Part 2 – a Review. Czech J. Food Sci., 25: 101–118.

This review article gives a survey of the biosynthetic pathways that lead to water-soluble vitamins in microorganisms,
plants and some animals. The biosynthetic pathways leading to some the B-group vitamins (biotin, folacin, cobalamins)
and to vitamin C are described in detail using reaction schemes and mechanisms with enzymes involved and detailed
explanations based on chemical principles and mechanisms.

Keywords: biosynthesis; B-group vitamins; biotin; folates; folic acid; cobalamins; vitamin B12; vitamin C; l-ascorbic acid;
d-erythro-ascorbic acid

The majority of water-soluble vitamins (thiamin,
riboflavin, pantothenic acid, vitamin B 6) and their
active forms were already reviewed (Velíšek &
Cejpek 2007).
6 Biotin
The biologically active (3aS,4S,6aR)-isomer of
biotin or [3aS-(3aα,4β,6aα)]hexahydro-2-oxo-
1H-thieno[3,4-d]imidazole-4-pentanoic acid, also
known as vitamin H (and formerly as bios II, factor
X, coenzyme R, or vitamin B 8), is required by all
living cells. It occurs as a prosthetic group of a
variety of enzymes known as carboxylases, trans-
carboxylases, and decarboxylases that catalyse
the transfer of carbon dioxide and thus play a
significant role in the biosynthesis of fatty acids
saccharides and in catabolism of branched-chain
amino acids.
Plants, fungi, and the majority of microorganisms
synthesise biotin and the pathway of its biosynthesis
seems to be conserved throughout the evolution
(Ploux 2000). This common pathway starts from
pimelic acid (Figure 20), which is first transformed

Partly supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project No. MSM 6046137305.

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Vol. 25, No. 3: 101–118 Czech J. Food Sci.

ATP + HS-CoA AMP + PP NH2

S N N
HOOC COOH CoA COOH H3C
EC 6.2.1.14 O S CH2 O N N

pimelic acid pimeloyl-CoA

L-alanine O
OH OH
COOH
EC 2.3.1.47
S-adenosyl-4-methylthio-2-oxobutanoic acid

HS-CoA + CO2 SAM

NH 2 NH 2

H 3C COOH H 3C COOH
O EC 2.6.1.62 NH2

8-amino-7-oxopelargonic acid
7,8-diaminopelargonic acid

ATP + CO2

EC 6.3.3.3

O L-methionine + NADPH + H S + SAM + NADP O ADP + P

HN NH HN NH

H H H H
HS H 3C
H
COOH
EC 2.8.1.6 COOH

dethiobiotin
NH 2

N
O N
H
1 3 5´
N N CH2
HN 2 NH O
3a +
H H
6a H 2´ 4´ 5´
6 5
S 4 COOH OH OH
1´ 3´
biotin 5´-deoxyadenosine

Figure 20
8
to the active form, pimeloyl-CoA 1) , by the action aminononanoic acid) by the action of 7,8-di-
of pimeloyl-CoA synthetase (6-carboxyhexanoyl- aminopelargonate transaminase (EC 2.6.1.62). De-
CoA ligase, EC 6.2.1.14). thiobiotin synthase (EC 6.3.3.3) then catalyses the
Under the catalysis by 8-amino-7-oxopelarg- formation of the imidazole part of dethiobiotin.
onate synthase (or 8-amino-7-oxononanonate The final unprecedented reaction in biochemis-
synthase, EC 2.3.1.47), pimeloyl-CoA reacts with try, the transformation of dethiobiotin to biotin
l-alanine and yields 8-amino-7-oxopelargonic acid is catalysed by biotin synthase (EC 2.8.1.6)9. The
(8-amino-7-oxononanoic acid), which is further biotin synthase Fe-S cluster functions as the im-
transformed to 7,8-diaminopelargonic acid (7,8-di- mediate sulfur donor for the biotin formation. This

8
For example, Bacillus subtilis requires free pimelic acid, but pimeloyl-CoA rather than free pimelic acid is required
by Escherichia coli.
9
This complex enzyme system comprises biotin synthase, l-cystein, dithioerythritol, Fe2+ ion, S-adenosyl-l-methionine
(SAM), and a reducing agent (flavodoxin, flavodoxin reductase, and NADP + or photoreduced deazaflavin). When
biotin synthase is reduced, the cluster dimerises to give a [4Fe-4S] + cluster, which is the catalytically important spe-
cies (Begley et al. 1999). Biotin is formed in substoichiometric amounts. Approximately three moles of SAM are
required per mole of biotin produced.

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

O OH O OH O
R ACP R ACP R ACP
S S S
OH
long-chain fatty acyl-ACP R H

O
H
O O O O
O O 1/2 O2
ACP R ACP
ACP H S S
HO S
O Fe
O
pimeloyl-ACP pimeloyl semialdehyde-ACP

Figure 21

reaction requires S-adenosyl-l-methionine (SAM)
which is reductively cleaved by the reduced Fe-S
cluster of biotin synthase to yield the adenosyl
radical. This radical then abstracts hydrogen from
the CH 3 group of dethiobiotin and the resulting
radical probably reacts with one of the sulfides of
the Fe-S cluster to yield the intermediate thiol,
and the abstraction of hydrogen from the first
CH 2 group in the side-chain then yields biotin.
The SAM derived products are l-methionine and
5’-deoxyadenosine (Begley et al. 1999).
Pimelic acid formation has been proposed to
proceed by oxidative cleavage of ACP-bound long-
chain fatty acid (Stok & De Voss 2000). The re-
action proceeds via two sequential hydroxylation
reactions to produce a diol intermediate. A third
oxidative reaction then produces either peroxide or
a peroxyl radical which subsequently decomposes
with C-C bond cleavage to yield two carbonyl
compounds, one of which is a pimeloyl semial-
dehyde derivative of ACP. The conversion of the
pimeloyl semialdehyde derivative to pimeloyl-ACP
may proceed either via autoxidation, cytochrome
P450 catalysed oxidation10, or both (Figure 21). The
acyl chain can be released as a free pimelic acid
e.g. by acyl-ACP hydrolase (acyl-ACP thioeste-
rase, EC 3.1.2.14), analogously to the fatty acids
formation (Velíšek & Cejpek 2006b).
vitamin M) (Friedrich 1988; Dewick 2002). Folic
acid, is a so called conjugate of 4-aminobenzoic
acid and l-glutamic acid, i.e. 4-[(pteridin-6-yl-
methyl)amino]benzoic acid attached to one or
more l-glutamic acid residues (IUPAC). Gener-
ally, three to eight glutamic acid residues occur in
natural folates. The active constituent of folates is
(6S)-5,6,7,8-tetrahydrofolic acid, i.e. (6S)-5,6,7,8-
tetrahydropteroylglutamic acid (H 4PteGlu).
The activity of tetrahydrofolic acid is analogous
to that of cobalamins as it functions as a carrier
of one-carbon functional groups which may be
in the form of methyl, methylene, methenyl, or
formyl groups. The vitamin is a coenzyme that
play a vital role in the metabolism of amino acids,
purine, and pyrimidine nucleotides.
Many microorganisms and plants synthesise
7,8-dihydrofolic (7,8-dihydropteroylglutamic)
acid (7,8-H 2PteGlu), the precursor of folic acid
coenzymes, de novo. Mammals, however, are de-
pendent for their nutrition on the basic molecule,
and can only carry out two step reduction of folic
acid to tetrahydrofolic acid via dihydrofolic acid
and the biosynthesis of dihydrofolic acid from folic
acid. Mammals can also add or remove additional
glutamyl residues.
7.1 7,8-Dihydrofolic acid

7 Folacin The biosynthesis of 7,8-dihydrofolic acid in mi-

croorganisms and plants starts from guanosine
Folacin is a common name for the biologically 5’-triphosphate (GTP), the common precursor
active derivatives of folic (pteroyglutamic) acid in the biosynthetic pathway to dihydrofolic acid
(formerly also known as vitamin B 9, vitamin B c or and to riboflavin (Friedrich 1988; KEGG) (Fig-

10
These proteins contain a cysteine-ligated heme with which they activate molecular oxygen, producing a molecule of water
and an equivalent of an Fe(V) oxo species that is responsible for the vast array of oxidative reactions that they perform. The
activation of oxygen requires two electrons, which are ultimately derived from NADH or NADPH and are usually supplied
by cytochrome P450 reductase (EC 1.6.2.4) in eucaryotes and an iron-sulfur redoxin (EC 1.8.4.8) in procaryotes.

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O O O
N NH2 NH2
HN HN HN

H2N N N 2 H2 O H COOH H 2N N O OH
NH H 2N N NH
PPPOCH2 PPPOCH2 PPPOCH2 N
O O OH HN OPPP
OH
EC 3.5.4.16 H 2N N N
H
OH OH OH OH OH O

guanosine 5´-triphosphate 2,5-diamino-4-oxo-6-(E-D-ribofuranosylamino)pyrimidine 7,8-dihydroneopterin 3´-triphosphat e

O
HO
H2O
H PP + P
glycolaldehyde
O AMP ATP O O OH
N N N
HN OPP HN OH HN OH
OH
H2N N N EC 2.7.6.3 H 2N N N EC 4.1.2.25 H2N N N
H H H

6-hydroxymethyl-7,8-dihydropterin diphosphate 6-hydroxymet hyl-7,8-dihydropterin 7,8-dihydroneopterin

4-aminobenzoic acid

EC 2.5.1.15
O COOH
PP H
COOH N COOH
O O N
n AT P + n Glu n AMP + nPP H
N N O COOH
HN N HN N n=0-6
H H
H2N N N H2N N N
H EC 6.3.2.12 H
7,8-dihydropteroic acid 7,8-dihydrofolic acid

Figure 22

ure 22). The cleavage of the imidazole ring of 6-Hydroxymethyl-7,8-dihydroneopterin diphos-

GTP and the elimination of the C-8 carbon as
formic acid is catalysed by GTP cyclohydrola-
se I (EC 3.5.4.16) and yields 2,5-diamino-4-oxo-
6-(ribofuranosylamino)pyrimidine as the first
intermediate. The elimination reaction is fol-
lowed by the Amadori rearrangement of thus
formed N-substituted β-d-ribofuranosylamine
to yield the corresponding Amadori product,
N-substituted 1-amino-1-deoxy-d-ribulose), and
then followed by ring closure of the pyrazine ring to
form 7,8-dihydroneopterin 3’-triphosphate (the com-
mon precursor for tetrahydrofolic acid, biopterin, and
many other natural pterins). Its dephosphorylation
by not yet characterised enzyme(s) yields 7,8-dihy-
droneopterin. The chain shortening of the side-chain
of 7,8-dihydroneopterin (elimination of glycolal-
dehyde by a retroaldolisation reaction), catalysed
by dihydroneopterin aldolase, EC 4.1.2.25) to form
6-hydroxymethyl-7,8-dihydroneopterin, is followed
by a reaction with ATP to form the corresponding
diphosphate (2-amino-4-hydroxy-6-hydroxymethyl-
dihydropteridine diphosphokinase, EC 2.7.6.3).
phate then reacts, under catalysis of dihydropteroate
synthase (EC 2.5.15), with 4-aminobenzoic acid to
form 7,8-dihydropteroic acid. The formation of 7,8-
dihydrofolic acid from 7,8-dihydropteroic acid and
l-glutamic acid, with participation of ATP, is cata-
lysed by dihydrofolate synthase (EC 6.3.2.12).
4-Aminobenzoic acid, forming part of the struc-
ture of 7,8-dihydrofolic acid, is produced from
chorismic acid (Figure 23). Chorismic acid is first
aminated to give 4-amino-4-deoxychorismic acid
(aminodeoxychorismate synthase/glutamine ami-
notransferase), which eliminates pyruvic acid (ami-
nodeoxychorismate lyase, EC 4.1.3.38) to yield
4-aminobenzoic acid.
7.2 Coenzyme forms of folic acid
The biosynthesis of the coenzyme forms of folic acid
consists of several steps, the reduction of dihydrofolic
acid to tetrahydrofolic acid (Figure 24) catalysed by
dihydrofolate reductase (EC 1.5.1.3), the incorpora-
tion and interconversion of C1 units of various kinds,

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

COOH L-glutamine L-glutamic acid COOH COOH

O
CH2 CH2
+
H3C COOH
O COOH EC 6.3.5.8 O COOH EC 4.1.3.38
OH NH 2 NH 2

chorismic acid 4-amino-4-deoxy chorismic acid 4-aminobenzoic acid p y ruvic acid

Figure 23

and the synthesis of the polyglutamyl chain. These
reactions are catalysed by a number of enzymes,
which have been studied in detail (KEGG).
8 Cobalamins
The term vitamin B 12 is used in two different
ways. In a broader sense, it refers to a group of
cobalt-containing organometallic compounds (the
covalent C-Co bond with cobalt(III) ion is the only
carbon-metal bond known in biochemistry). These
compounds contain 5,6-dimethylbenzimidazole
and are known as cobalamins. The most impor-
tant cobalamins are cyanocobalamin (vitamin
B12), the intermediate forms of the cofactors, i.e.
aquacobalamin (also known as vitamin B 12a), and
hydroxocobalamin (vitamin B12b, aquacobalamin
is the conjugate acid of hydroxocobalamin), and
the two metabolically active coenzyme forms of
vitamin B12, methylcobalamin (methylvitamin B12)
and 5'-deoxy-5'-adenosylcobalamin (often called
5'-deoxyadenosylcobalamin or adenosylvitamin B12).
In a more specific way, the term vitamin B12 is used
to refer to only one of these forms, cyanocobalamin,
which is the principal vitamin B 12 form used for
foods and in nutritional supplements (IUPAC).
Cobalamins belong to a group of compounds
called corrinoids that are based upon the skeleton
of corrin (Figure 25). The corrin nucleus con-
tains four partially reduced pyrrole rings (A, B,
C, and D) joined into a macrocyclic-ring system
by links between their α position. Three of these
links are formed by one-carbon unit (methine
bridge) and the other by a direct Cα-Cα bond
between rings A and D. This highly substituted
macrocycle is adorned with seven peripheral me-
thyl groups. Attached to the corrin ring are seven
amide chains, named a-g. On the side α axial of the
pseudo-planar framework is a distinct nucleotide
loop, containing a 2,3-dimethylimidazole that
coordinates cobalt in 1-position and continues as
a nucleotide-like moiety from the 3-position, i.e.
with a ribose ring, a phosphate and eventually an
N-propyl propanamide linkage back to the corrin
ring at the f-chain. The β axial ligand of the corrin
ring can be a variety of neutral species and anions.
The species of known biochemical occurrence
include 5'-deoxy-5'-adenosylcobalamin (R = 5'-de-

O COOH
H
N COOH
O N
H
N O COOH
HN N n = 0-8
H
H2N N N
H 7,8-dihydrofolic acid

NADPH + H

EC 1.5.1.3

NADP O COOH
6´ H
1´ N COOH
5´
O 7´ N  
H 9 H 
4a N 10 2´ O COOH
H N3 4 5 6 N 4´ n = 0-8
H 3´
2 1
8 7
H 2N N 8a N
H (S)-5,6,7,8-tetrahydropteroic acid

Figure 24

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Vol. 25, No. 3: 101–118 Czech J. Food Sci.

b
3 3
4 4
5 5
6
7
6
7 32 34 c
CONH2 38 40
22 AA B 8 33
a
B 8 31 CONH 2
35 39
N N
1
1 N N99 29 27 H C
25 30 CH3 37 36
CH3 42
d
21 22 10 H 2NO C 3
44
19 10 28
NH24 N 23 11 CONH2
18
19D
NH CN 12 11 26
20
21
N
R  N 22 41 43 45
D C 12 H 3C
18 17 16 15 14 13 Co (III)
17 16
15
14 13 g 60 N  N 47
24 23 CH3
63 61
H 2NO C 54
corrin 62 46 CH3
H 3C
53 CH3 48
56 55
49
51
e
5759
CONH
A10 58 O NH Pr3 50 52 2 B10
NH 2 f Pr1
B3
CH3 N
B9
B4
B5 CH
3

A7 N A5 Pr2 B2
A6 N A1
O O4 B7 B11
A8 O3 O O5 N B8 B6 CH3
A15 A16 A2 P OH B1
R= A9 N
A4 N
O O2 O R7 OH
A14 A11 A3
R3 R2
A13 A12 R4 R1
OH OH R8 R5 O
A18 A17 HO R6

5´-deoxy-5´-adenosyl 5´-deoxy-5´-adenosylcobalamin

Figure 25

oxy-5'-adenosyl), methylcobalamin (R = CH3), aqua-
cobalamin (R = H2O), cyanocobalamin (R = CN-),
with the ligands coordinated to cobalt (Sirovatka
et al. 2000; Jensen & Mikkelsen 2001; Marques
et al. 2001).
There is a close structural relationship between
corrinoids and porphyrins (primarily represented
by chlorophylls and heme pigments). However,
one of the most prominent features of corrinoids
is that the macrocycle has undergone a unique
extrusion of one of the ring carbons (C-20) during
the process of the ring contraction. The numbering
of corrinoids is thus the same as that of porphyrin
nucleus, number 20 being omitted to preserve the
identity (Figure 25).
In spite of their apparently similar structures, the
B12 coenzymes function as cofactors for two quite
different types of enzymatic reactions ( Jensen &
Mikkelsen 2001; Marques et al. 2001; Marsh
& Drennan 2001). Methylcobalamin is used in
certain transmethylation reactions, including
methylation of homocysteine to form methionine
and in the pathway for fixing carbon dioxide in
certain anaerobic acetogenic microorganisms. All
methylcobalamin-dependent enzymes transfer a
methyl cation by heterolytic cleavage of the co-
balt-carbon bond, giving the Co(I) intermediate.
The 5'-deoxy-5'-adenosylcobalamin-dependent
enzymes, on the other hand, generate a substrate
radical and the Co(II) square-pyramidal interme-
diate, induced by homolytic cleavage of the Co-C
bond (the so-called activation of the coenzyme,
which is the first step in the catalytic cycle). The
cofactor is involved in the enzymatic catalysis of

HOOC

NH2

HS-CoA + ATP ADP + P glycine HS CoA CO 2

O O O
HOOC CoA HOOC H2 N
COOH S COOH COOH COOH
EC 6.2.1.5 EC 2.3.1.37 NH2 EC 2.3.1.37

succinic acid succinyl-CoA

Figure 26L-2-amino-3-oxoadipic acid 5-aminolevulinic acid

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

COOH COOH
COOH COOH

COOH H 2O 4 NH 3 H 2O
HOOC HOOC HOOC
COOH COOH
NH HN NH HN
4 HO
H 2N
N EC 2.5.1.61 EC 4.2.1.75
H NH HN NH HN
HOOC COOH HOOC COOH
porphobilinogen

8 H 2O HOOC
EC 4.2.1.24 COOH COOH COOH

O hydroxymethylbilane uroporphyrinogen III

8 H2N SAM
COOH

5-aminolevulinic acid EC 2.1.1.107

SAH
COOH COOH COOH
COOH COOH COOH

CH3 H 3C CH3 H3C

H 3C HOOC HOOC
HOOC COOH COOH COOH
N HN N HN N N
SAH SAM SAH SAM
H3C
NH HN NH HN
NH HN

HOOC COOH HOOC COOH HOOC COOH

EC 2.1.1.130 EC 2.1.1.107

COOH COOH COOH COOH COOH COOH

precorrin 3A precorrin 2 precorrin 1

NADH + H + O2

EC 1.14.13.83

NAD + H2O

COOH COOH COOH

COOH COOH
COOH
H 3C CH3 H 3C CH3
H 3C CH3 HOOC HOOC
COOH COOH COOH
NH N H3C NH N H3C NH N
O O
H 3C SAM SAH
SAM SAH O O
HO CH3
NH HN N HN N N

HOOC COOH HOOC COOH HOOC COOH

H3C EC 2.1.1.133 H3C
EC 2.1.1.131

COOH COOH COOH COOH COOH COOH

precorrin 3B precorrin 4 precorrin 5

SAM + H2O

EC 2.1.1.152

SAH + CH3COOH

COOH COOH
COOH
COOH COOH
COOH
CH3 H 3C H 3C CH3
H 3C CH3 HOOC CH3 HOOC
HOOC COOH
COOH COOH
NH N NH N
N N H3C H 3C
H3C NADP + H2O NADPH + H
2 SAH + CO2 2 SAM
CH3 CH3
CH3 N N N HN
N N
CH3
HOOC COOH HOOC COOH
HOOC
H 3C H 3C
H 3C
CH3 EC 2.1.1.132 EC 1.3.1.54

COOH COOH COOH COOH

COOH COOH
precorrin 8X precorrin 6B precorrin 6A

EC 5.4.1.2

COOH COOH COOH

COOH CONH2 CONH2
CH3 CH3 CH3
H 3C CH3 H 3C CH3 H 3C CH3
HOOC H 2NO C H 2NO C
COOH COOH COOH
NH N NH N N N
H 3C H3C H3C
2 Co
2 ATP + 2 Gln + 2 H2O 2 ADP + 2 P + 2 Glu ATP + Co ADP + P + H
N N N N N N
CH3 CH3 CH3
HOOC HOOC HOOC
CH3 CH3 CH3
H3C EC 6.3.5.9 H 3C H 3C
CH3 CH3 EC 6.6.1.2 CH3

COOH COOH COOH COOH COOH COOH

hydrogenobyrinic acid hydrogenobyrinic acid a,c diamide cob(II)yrinic acid a,c diamide

Figure 27

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Vol. 25, No. 3: 101–118 Czech J. Food Sci.

a variety of chemically difficult intramolecular 3-oxoadipic (l-2-amino-3-oxohexanedioic) acid,

1,2-rearrangements catalysed by 5'-deoxy-5'-ade-
nosylcobalamin-dependent isomerases and in the
reduction of ribonucleotides to deoxyribonucle-
otides in some organisms.
Vitamin B12 is synthesised by many bacteria, some
cyanobacteria and even some yeasts. The ultimate
source of cobalamin for mammalian metabolism
is microbial synthesis. The cobalamins from the
microbial flora in the gastrointestinal system of
herbivorous animals are absorbed by the host and
stored in their tissues. From there, cobalamin is
passed on to other animals in the food chain.
The biosynthesis of cobalamin by bacteria pro-
ceeds either aerobically or anaerobically (IUPAC;
KEG G; Roessner et al. 2001). The starting
compound of both pathways is 5-aminolevulinic
(5-amino-4-oxopentanoic) acid derived from the
glycine metabolism. The condensation of glycine
with succinyl-CoA is catalysed by 5-aminolevuli-
nate synthetase (EC 2.3.1.37) and yields l-2-amino-
which eliminates carbon dioxide to produce 5-ami-
nolevulinic acid. In the presence of Mg 2+ and ATP,
the biosynthesis of succinyl-CoA from succinic
acid is catalysed by succinate-CoA ligase (ADP-
forming) (EC 6.2.1.5) also known as succinate
thiokinase (Figure 26).
The first three steps of both pathways (aerobic and
anaerobic) follow those of porphyrin biosynthesis
(Figure 27). These steps start by the condensation
of two 5-aminolevulinic acids to form porpho-
bilinogen (aminolevulinic acid dehydratase, also
known as porphobilinogen synthase, EC 4.2.1.24).
This reaction is followed by the conversion of four
porphobilinogens to yield hydroxymethylbilane
(hydroxymethylbilane synthase, EC 2.5.1.61) 11. In
the presence of a second enzyme, uroporphyrino-
gen III synthase (EC 4.2.1.75), which is often called
cosynthase, hydroxymethylbilane is cyclised under
the ring D inversion to form uroporphyrinogen III
(urogen III), the common intermediate in the biosyn-

COOH COOH
COOH
HOOC HOOC
HOOC

HOOC HOOC COOH

COOH HOOC COOH
NH HN NH HN
NH HN
HO H2O

NH HN NH HN
HN
HN
HOOC COOH HOOC H2C COOH
COOH

EC 4.2.1.75 COOH
HOOC
COOH COOH COOH
COOH
HOOC
hydroxymethylbilane

COOH COOH
HOOC HOOC

H
HOOC COOH HOOC COOH
NH HN NH HN

NH HN NH HN
HOOC COOH HOOC COOH
H

COOH COOH COOH COOH

uroporphyrinogen III

Figure 28

11
The enzyme contains at its active site a unique dipyrrolomethane cofactor on which the tetrapyrrole is built. The
enzyme works by stepwise addition of pyrrolylmethyl groups until a hexapyrrole is present at the active centre.
The terminal tetrapyrrole is then hydrolysed to yield the product, leaving a cysteine-bound dipyrrole on which the
assembly continues.

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

COOH

H 3C

HOOC N
H
H3C
Fe (III) O NH COOH COOH COOH

HOOC

H3C H 3C H 3C
SAH HOOC

precorrin 3A O NH O NH H3C NH
COOH O O
HO H3C
H O
EC 1.14.13.83 H3C H O
NH N EC 2.1.1.131 N
HOOC HOOC HOOC
H3C
COOH CH3
HOOC S
COOH Ado COOH COOH
H3C NH2

HOOC NH SAM precorrin 3B precorrin 4

O
H 3C
NH

HOOC

COOH

Figure 29

thesis of the corrinoids and the porphyrins (Figure
28). If uroporphyrinogen III synthase is absent,
hydroxymethylbilane cyclises spontaneously. The
next step is catalysed by uroporphyrin III methyl-
transferase (EC 2.1.1.107). The enzyme catalyses two
sequential methylation reactions, the first forming
precorrin 1 by methylation of C-2 carbon, which is
further methylated at C-7 to form precorrin 2. Up
to this point the aerobic and anaerobic pathways
are virtually undistinguishable.
In the aerobic cobalamin biosynthesis (e.g. by
Pseudomonas denitrificans), the next product
formed by methylation of precorrin 2 at C-20 is
precorrin 3A (precorrin 2 C-20-methyltransferase,
EC 2.1.1.130) (Figure 27). The subsequent reaction
(an oxygen atom from oxygen is incorporated into
the macrocycle at C-20 as a hydroxyl group and a
γ-lactone is formed; this hydroxylation spring-loads
the mechanism for the contraction of the macro-
cycle by installing a masked pinacol) is carried out
by precorrin 3B synthase (EC 1.14.13.83), yielding
precorrin 3B (precorrin 3 hydroxylactone) as the
product. The mechanism of hydroxylation at C-20
of precorrin 3A with the subsequent formation of
a γ-lactone between C-1 and the acetate of ring
A is explained by either the direct insertion of
oxygen from an Fe(III)-O+ species or by the forma-
tion of a 1,20-epoxide, which, for steric reasons,
requires ring opening through the participation of
the nitrogen electron pair rather than by a direct
attack by the carboxylate of the C-2 acetate side
chain (Figure 29). The formation of precorrin 3B
is followed by three methylation reactions which
introduce methyl groups at C-17 (precorrin 3B
C-17-methyltransferase, EC 2.1.1.131, Figures
27 and 29) 12 , C-11 (precorrin 4 C-11-methyl-
transferase, EC 2.1.1.133), and C-1 (precorrin
6A synthase, deacetylating, EC 2.1.1.152) of the
macrocycle followed by acetic acid extrusion,
giving rise to precorrin 4, precorrin 5 and pre-

12
The addition of the fourth methyl group at C-17 to afford precorrin 4, catalysed by precorrin 3B C17-methyltrans-
ferase (EC 2.1.1.131), triggers the ring contraction, and is accompanied by the formation of a methyl ketone pendant
from C-1 that has been derived by a pinacol-type rearrangement.

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Vol. 25, No. 3: 101–118 Czech J. Food Sci.

COOH COOH
COOH COOH

H3C CH3 H 3C CH3

HOOC HOOC
COOH COOH
NH N HO NH N
H 3C H3C

O CH3 O CH3
N N EC 2.1.1.152 N N

HOOC COOH HOOC COOH

H H 3C H 3C
H

COOH COOH COOH COOH

precorrin 5
H 3C COOH

COOH COOH
COOH COOH

H 3C CH3 SAH H3C CH3

HOOC HOOC
COOH COOH
Ado
NH N NH N
H 3C S CH3
CH3 CH3
N HN N N

HOOC COOH H 2N HOOC H COOH

H 3C COOH H 3C

SAM
COOH COOH COOH COOH

precorrin 6A

Figure 30

corrin 6A, respectively (Figure 30)13. Precorrin A
reductase (EC 1.3.1.54) then catalyses the forma-
tion of precorrin 6B (dihydroprecorrin 6) from
precorrin 6A (C-18/C-19 reduction) (Figure 31).
This reaction is followed by the formation of pre-
corrin 8 also known as precorrin 8X (C-5 and C-15
methylation and decarboxylation) using precorrin
6Y C-5,-15-methyltransferase (decarboxylating)
(EC 2.1.1.132). A rearrangement of the C-11 me-
thyl group (to become the C-12 re-methyl group
found in vitamin B12) then yields hydrogenobyrinic
acid (precorrin 8X methylmutase, EC 5.4.1.2). The
next step in the aerobic biosynthesis of cobalamin,
a,c-amidation, generates hydrogenobyrinic acid
a,c diamide (hydrogenobyrinic acid a,c diamide
synthase, EC 6.3.5.9), the substrate required by
cobaltochelatase (EC 6.6.1.2), which adds cobalt
(Co2+ ion) to the macrocycle yielding cob(II)yrinic
acid a,c diamide.
The first difference between the biosynthesis
of vitamin B 12 in aerobic and anaerobic condi-
tions is that the anaerobes (e.g. Salmonella ty-
phimurium and Propionibacterium shermanii)
synthesise, in an NAD + -dependent dehydro-
genation, sirohydrochlorin (the precursor of
the pigment siroheme in methanogenic bacteria
formed under the catalysis of sirohydrochlorin
ferrochelatase, EC 4.99.1.4) from precorrin 2 in-
stead of precorrin 3 (precorrin 2 dehydrogenase,
EC 1.3.1.76), and then the cobalt atom is inserted
at this stage (sirohydrochlorin cobaltochelatase,
EC 4.99.1.3) to yield cobalt precorrin 2 (Co-pre-
corrin 2 or Co-sirohydrochlorin) (Figure 32).
Methylation at C-20 of Co-precorrin 2 yields Co-
precorrin 3 (precorrin 2 C-20-methyltransferase,
EC 2.11.130). Methylation of Co-precorrin 3 at
C-17, followed by the ring contraction and δ-lac-
tone formation (precorrin 3B C-17-methyltrans-

13
Precorrin 6A synthase (EC 2.1.1.152) is a bifunctional enzyme that catalyses the removal of the methyl ketone moiety
of a tautomer of precorrin 5 (deacetylating enzyme). Deacetylation provides an intermediate that then undergoes
C-1 methylation to afford a tautomer of precorrin 6A, which finally isomerises to precorrin 6A.

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

NH 2
Ado
S COOH
COOH CH3 COOH
SAM
COOH COOH
2 SAH + CO2 H3C H
H3C CH3 H 3C CH3
HOOC HOOC
COOH COOH
N N N N
H 3C H H3C
O H
H
CH3 CH3
N N O H EC 2.1.1.132 N N
CH2
HOOC HOOC
H
H 3C H 3C
precorrin 6B CH3

COOH CH3 COOH COOH COOH

S COOH
Ado

SAM NH 2

COOH
COOH
CH3
H 3C CH3
HOOC
COOH
N N
H3C

CH3
N N
CH3
HOOC
H 3C
CH3

COOH COOH

precorrin 8X

Figure 31

ferase, EC 2.1.1.131) yields Co-precorrin 4 which
is methylated at C-11 (precorrin 4 C-11-methyl-
transferase, EC 2.1.1.133) to form Co-precorrin 5.
Methylation at C-1 of Co-precorrin 5, followed
by acetaldehyde extrusion (methyltransferase,
EC 2.1.1.-), yields Co-precorrin 6A (Figure 33),
which is reduced at C-18/C-19 (precorrin 6A re-
ductase, EC 1.3.1.54) to Co-precorrin 6B, and this
reaction is followed by C-5 and C-15 methylation
and decarboxylation (precorrin 6Y C-5, -15-meth-
yltransferase, EC 2.1.1.132) to yield Co-precorrin
8X. The C-11 methyl rearrangement (precorrin
8X methylmutase, EC 5.4.1.2) produces cobyrinic
acid, which is finally amidated by an amide syn-
thase (EC 6.3.1.-) to produce cob(II)yrinic acid
a,c-diamide.
Cob(II)yrinic acid a,c diamide is the product
from which the two pathways converge so that the
next steps are the same in both aerobes and anaer-
obes (Figure 34). This compound is first reduced
to a Co 1+ complex, cob(I)yrinic acid a,c diamide,
under the action of cob(II)yrinic acid a,c diamide
reductase (EC 1.16.8.1). The reduction is followed
by adenosylation of cob(I)yrinic acid a,c diamide
catalysed by cob(I)yrinic acid a,c diamide 5'-deoxy-
5'-adenosyltransferase (EC 2.5.1.17), which yields
adenosylcobyrinic acid a,c-diamide containing
Co(III). 5'-Deoxy-5'-adenosylcobyric acid synthase
(EC 6.3.5.10) then catalyses the four-step amida-
tion sequence from cobyrinic acid a,c-diamide to
5'-deoxy-5'-adenosylcobyrinic acid hexaamide,
known as 5'-deoxy-5'-adenosylcobyric acid, via
the formation of cobyrinic acid triamide, tetra-
amide, and pentaamide intermediates. The same
enzyme also catalyses the attachment of (R)-1-ami-
nopropan-2-ol to 5'-deoxy-5'-adenosylcobyric
acid. The substrate for this reaction, (R)-1-amino-
propan-2-yl phosphate, is produced by threonine
phosphate decarboxylase (EC 4.1.1.81). The enzyme
that converts l-threonine to O-phospho-l-threo-
nine (l-threonine phosphate) is not yet character-
ised. Adenosylcobinamide kinase (EC 2.7.1.156),
adenosylcobinamide phosphate guanylyltrans-
ferase (EC 2.7.7.62), and cobalamin synthase
(EC 2.7.8.26) catalyse reactions in the nucleotide
loop assembly pathway, which convert 5'-deoxy-
5'-adenosylcobinamide into 5'-deoxy-5'-ade-
nosylcobalamin, vitamin B 12 prosthetic group.

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Vol. 25, No. 3: 101–118 Czech J. Food Sci.

COOH COOH COOH

COOH COOH COOH

H 3C CH3 H3C CH3 H3C CH3

HOOC COOH HOOC COOH HOOC COOH
N HN N H N N N
2 Co
NAD NADH + H Co 2H
NH HN N
H N N N

HOOC COOH HOOC COOH HOOC COOH

EC 1.3.1.76 EC 4.99.1.3

COOH COOH COOH COOH COOH COOH

precorrin 2 sirohydrochlorin Co-precorrin 2

Figure 32

Nicotinate mononucleotide-dimethylimidazole 9 Vitamin C

phosphoribosyltransferase (EC 2.4.2.21) and α-ri-
bazole phosphatase (EC 3.1.3.73) are involved in Vitamin C is the collective term that comprises l-as-
5,6-dimethylbenzimidazole activation whereby corbic acid, the product of its one-electron oxida-
5,6-dimethylbenz­imidazole is converted to its ri- tion, l-ascorbyl radical or l-monodehydroascorbic
boside, α-ribazole, N1-(α-d-ribofuranosyl)-5,6-di- acid or l-semidehydroascorbic acid, and the product
methylbenzimidazole (Figure 35). The second of its two-electron oxidation, l-dehydroascorbic
branch of the nucleotide loop assembly pathway acid (Velíšek 2002). Plant-related functional roles
is the cobinamide activation branch where 5'-de- involving l-ascorbic acid are largely related to its
oxy-5'-adenosylcobinamide or 5'-deoxy-5'-ad- redox properties. It has a role(s) in photosynthesis
enosylcobinamide phosphate is converted to the and plant stress through the control of active oxygen,
activated intermediate 5'-deoxy-5'-adenosylcob- in cell wall growth and development, and possibly
inamide-GDP. The final step in 5'-deoxy-5'-ade- also in cell division. Chain cleavage of l-ascorbic
nosylcobalamin biosynthesis is the condensation acid by hydrogen peroxide may have a major role
of adenosylcobinamide-GDP with α-ribazole to in plant metabolism as it results in specific end
yield 5'-deoxy-5'-adenosylcobalamin 14. products such as l-threonic acid, l-tartaric acid,

COOH COOH COOH

C OOH COOH
COOH

H3C CH3 H3C CH3 H3C CH3

HO HOOC
COOH C OOH COOH
O O
N N N N N N
O H 3C
Co O Co
SAM SAH Co SAM + H2O SAH + CH3CH=O
H 3C H 3C CH3
N N N N CH3 N N

HOOC C OOH HOOC COOH HOOC COOH

H3C H3C H3C
EC 2.1.1.133 EC 2.1.1.152

C OOH COOH COOH COOH COOH COOH

Co-precorrin 4 Co-precorrin 5 Co-precorrin 6A

Figure 33

14
In the corrinoid adenosylation pathway, reduction of Co(III) to Co(II) in aquacob(III)alamin (vitamin B 12a) proceeds by a
one-electron transfer. This can be carried out by an NADH-linked flavoprotein aquacobalamin reductase (EC 1.16.1.3) or
non-enzymatically in the presence of dihydroflavin nucleotides, to yield vitamin B12r. In the next reaction, Co(II) is reduced
to Co(I) in a second single-electron transfer by cob(II)alamin reductase (EC 1.16.1.4), and the cob(I)alamin (vitamin B 12s)
conducts a nucleophilic attack on the adenosyl moiety of ATP to leave the cobalt atom in a Co(III) state and yield vitamin
B12 coenzyme. The reaction is catalysed by cob(I)yrinic acid a,c-diamide adenosyltransferase (EC 2.5.1.17). A flavoprotein
cyanocobalamin reductase (EC 1.16.1.6) catalyses the formation of cob(I)alamin from cyanocob(III)alamin (vitamin B 12).
The formation of methylcobalamin (methylvitamin B 12) probably occurs in the process of the complex methyltransferase
reaction catalysed by N5-methyltetrahydrofolate homocysteine methyltransferase (EC 2.1.1.13).

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

COOH COOH COOH

CONH2 CONH2 CONH 2
CH3 CH3 C H3
H3C CH3 H3C CH3 H3C C H3 NH2
H2NO C H2NO C H2NO C
COOH COOH C OOH N
N N N N N
H3C H3C N N
H3C
Co Co Co O N N
1/2 FMN 1/2 FMNH 2 ATP + H2O P + PP
N N N N N N
C H3 CH3 CH3
HOOC HOOC HOOC
CH3 CH3 CH3
H3C H3C H3C OH OH
CH3 CH3 EC 2.5.1.17 C H3
EC 1.16.8.1

COOH COOH COOH COOH COOH COOH

cob(II)yrinic acid a,c diamide cob(I)yrinic acid a,c diamide 5´-deoxy-5 ´-adenosylcobyrinic acid a,c diamide

OH OP
COOH COOH 4 Gln + 4 ATP + 4 H 2O
H3C H3C
NH2 NH2 EC 6.3.5.10
CONH2 L-threonine O-phospho-L-threonine
CONH2 4 Glu + 4 ADP + 4 P
CH3
H3C CH3 NH2
H2NO C EC 4.1.1.81
CONH2 N C O2 C ONH2
N
N N CONH 2
H3C
N OP
Co O N C H3
NH2
NH2 H3C C H3
H2NO C
N N H3C C ONH2 N
C H3 N
H2NO C N N
CH3 (R)-1-aminopropan-2-yl H3C
H3C OH OH N
CH3 ADP + P phosphate + ATP Co O N
N N
O CH3 C ONH2 CH3
N H2NO C
H CH3 OH OH
OH 5´-deoxy-5´-adenosylcobinamide H3C
ATP
EC 6.3.1.10 C H3

EC 2.7.1.156 COOH CONH2

5´-deoxy-5´-adenosylcobyric acid
ADP
CONH2
CONH2
CONH2
GTP PP C ONH2
CH3
H3C CH3 NH2 CH3
H2NO C H 3C CH3 NH2
CONH2 N H2NO C
N CONH2 N
N N N
H3C N N
Co O N N EC 2.7.7.62 H3C
Co O N N
N N
C H3 N N
H2NO C C ONH2 C H3
CH3 OH OH H2NO C
H3C CONH 2 CH3
CH3 H3C OH OH
C H3 CH3
H3C CH3 NH2 O
CH3 C ONH2 H2NO C
O N C ONH2 N CH3 C ONH2
N O N
H N N N NH
OP H3C H
Co N O P P O
O N N N NH2
5´-deoxy-5 ´-adenosylcobinamide phosphate O
N N
CH3
H2NO C
CH3 OH OH
H3C OH OH
C H3 EC 2.7.8.26
5´-deoxy-5´-adenosylcobinamide-GDP
CONH2
O NH
N C H3 CH3
C H3 N
GMP
O N C H3 N CH3
P
O OH OH OH

O O
HO HO
vitamin B12 coenzyme (5 ´-deoxy-5´-adenosylcobalamin) -ribazole

Figure 34

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Vol. 25, No. 3: 101–118 Czech J. Food Sci.

riboflavin COOH
+ H
N N CH3 N CH3
COOH nicotinic acid
N CH3 N CH3
H2O P
OH OH OH OH
PO N
N CH3 O
+
N CH3 EC 2.4.2.21 O EC 3.1.3.73 O
H PO HO
OH OH

5,6-dimethylbenzimidazole nicotinic acid D-ribonucleotide -ribazole 5´-phosphate -ribazole

Figure 35

l-glyceric acid, oxalic acid, and CO2, or is recycled
through triose and hexose phosphates. In animals,
l-ascorbic acid is essential for the formation of
collagen, the principal structural protein in skin,
bone, tendons, and ligaments, being a cofactor in the
hydroxylation of l-proline to (E)-4-hydroxy-l-pro-
line and of l-lysine to 5-hydroxy-l-lysine (Dewick
2002; Velíšek & Cejpek 2006a). Ascorbic acid is
also associated with the hydroxylation of l-tyrosine
in the pathway to catecholamines (Velíšek et al.
2006), in the biosynthesis of homogentisic acid,
and the precursor of tocopherols and plastoqui-
nones. It is best known as an antioxidant that can
detoxify reactive products of oxygen metabolism,
e.g. superoxide radical, hydrogen peroxide, and
singlet oxygen (Velíšek 2002).
The biosynthesis of l-ascorbic acid occurs by
different pathways in plants and animals. It is bio-
synthesised in all chlorophyll-containing plants.
Among animals, this ability is absent only in some
of the more primitive forms (i.e. in the insects
and invertebrates), most fish and a few avian and
mammalian species including humans. The ability
to synthesise ascorbic acid begins in the amphib-
ians, and is located in the kidneys in this class, as
well as in reptiles, more primitive orders of birds,
and egg-laying mammals. Many marsupials use
both their kidneys and livers for this synthesis.
The higher orders of birds and most mammals
synthesise ascorbic acid in the liver. The most
recent orders of birds, as well as several mam-
mals (among others guinea pigs, bats, and some
primates including humans), have lost their abil-
ity to synthesise ascorbic acid and must ingest
exogenous ascorbic acid in their food.
9.1 l-Ascorbic acid
9.1.1 Biosynthesis in animals
In animals, l-ascorbic acid is formed from the active
form of d-glucose (Smirnoff 2001), UDP-d-glu-
cose (UDP-d-Glc), as a part of the UDP-glucose
interconversion pathway. 15 UDP-d-Glc is oxi-
dised at C-6 by UDP-d-glucose dehydrogenase
(EC 1.1.1.22), forming irreversibly UDP-d-glu-
curonic acid (UDP-d-GlcA) (Figure 36). UDP-d-
GlcA is then transformed to UDP-d-glucuronic acid
1-phosphate (UDP-D-GlcA1P) which is hydrolysed
releasing d-GlcA or d-glucurono-3,6-lactone. The
intermediates and enzymes, which could include
(pyro)phosphorylases and (pyro)phosphatases (e.g.
UDP-glucuronic acid pyrophosphatase and glu-
curonic acid 1-phosphate phosphatase), involved in
these steps, do not appear to have been identified.
There is apparently no information on these en-
zymes. Non-specific lysosomal acid phosphatases
could account for some of the activity. Reduc-
tion at C-1 of d-GlcA by d-glucuronate reductase
(EC 3.1.1.19) or of d-glucurono-3,6-lactone by
d-glucuronolactone reductase (EC 3.1.1.20) then
yields l-gulonic acid (l-GulA)16 or l-gulono-1,4-lac-
tone (l-xylo-hexulono-1,4-lactone). Moreover,

15
The starting point of the biosynthetic pathway can be considered d-fructose 6-phosphate (d-Fru6P), which reversibly
isomerises to d-glucose 6-phosphate (d-Glc6P) by phosphoglucose isomerase (EC 5.3.1.9). Transformation of d-Glc6P
to d-glucose 1-phosphate (d-Glc1P) is a reaction catalysed by phosphoglucomutase (EC 5.4.2.2). UDP-d-glucose
(UDP-d-Glc) is formed from d-Glc1P and uridine 5'-triphosphate (UTP) in a reaction catalysed by UDP-d-glucose
pyrophosphorylase (EC 2.7.7.9). UDP-d-Glc can also arise from UDP-d-Gal via an epimerisation reaction involving
UDP-d-glucose 4-epimerase (EC 5.1.3.2) (Velíšek & Cejpek 2005).
16
The enzyme EC 1.1.1.19 also reduces d-galacturonic acid. May be identical with EC 1.1.1.2. The enzyme EC 3.1.1.25
is specific for 1,4-lactones with 4-8 carbon atoms (requires Ca 2+). The enzyme EC 1.1.3.8 is a flavoprotein (FAD).

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

2 NAD + H2O 2 NADH + 2 H H 2O UMP

CH2 OH COOH COOH
O O O
OH OH OH
sp eculative
HO OPPU EC 1.1.1.22 HO OPPU HO OP
OH OH OH
UDP-D-glucose UDP-D-glucuronic acid D-glucuronic acid 1-phosphate
H 2O

6 P
O
H 56 COOH
O EC 3.1.1.19 O
HO
4 1 OH
O
2
OH HO OH
3
OH OH
H 2O
D-glucurono-6,3-lactone D-glucuronic acid
NADPH + H NADPH + H

EC 1.1.1.20 EC 3.1.1.19
NADP NADP
1
CH2 OH COOH
H 2O
H OH HO H
O
HO H
OH HO O
H OH
EC 3.1.1.25
HO H
L-gulono-1,4-lactone
CH2 OH
1/2 O2
L-gulonic acid
EC 1.1.3.8

H 2O
CH2 OH CH2 OH
H OH H OH
O O
OH O O

O OH OH
L-2-oxogulono-1,4-lactone L-ascorbic acid
Figure 36

l-GulA can be transformed to l-gulono-1,4-lac- 9.1.2 Biosynthesis in plants

tone by the action of 1,4-lactonase (EC 3.1.1.25).
l-Gulono-1,4-lactone is oxidised at C-2 to l-2-ox-
ogulono-1,4-lactone (l-xylo-hex-2-ulosono-1,4-lac-
tone) by l-gulonolactone oxidase (EC 1.1.3.8), and
spontaneously isomerises to l-ascorbic acid. During
the course of these transformations, an apparent
inversion of configuration occurs so that the C-1
of d-Glc is incorporated into the C-6 of l-ascorbic
acid molecule (inversion of the hexose chain).
Surprisingly, the biosynthetic pathway of l-as-
corbic acid in plants was not elucidated un-
til relatively recently ( Wheeler et al. 1998).
Plants form l-ascorbic acid from GDP-d-man-
nose (GDP-d-Man). 17 The proposed reaction
sequence (Figure 37) involves the oxidation of
the C-4 hydroxyl group to a carbonyl group,
w ater elimination, the re addition of w ater
from the opposite face of the double bond, and

17
The starting point of the biosynthetic pathway in plant can be considered to be d-Fru6P which reversibly isomerises to
d-mannose 6-phosphate (d-Man6P) (under the catalysis by phosphomannose isomerase, EC 5.4.2.8) and d-Man6P to
d-mannose 1-phosphate (d-Man1P) (phosphomannomutase, EC 5.4.2.8). d-Man1P then reacts with guanosine 5'-triphos-
phate (GTP) to yield GDP-d-mannose (GDP-mannose pyrophosphorylase, EC 2.7.7.22) (Velíšek & Cejpek 2005).

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Vol. 25, No. 3: 101–118 Czech J. Food Sci.

CH2 OH
O O
CH2 OH
HO HO
HO OPPG EC 5.1.3.18 HO OPPG
OH OH

GDP-D-mannose GDP-L-galactose

H2O (hy drolase)

GMP
CH2 OH NAD NADH + H P
H OH
O O O
CH2 OH CH2 OH
HO O HO
HO
EC 1.1.1.48 OH EC 3.1.3.23 OP
HO HO
OH OH OH
L-galactono-1,4-lactone L-galactose L-galactose 1-phosphate
3
2 Fe

EC 1.3.2.3
2
2 Fe
CH2 OH CH2 OH
H OH H OH
O O
O O

OH O OH OH
L-2-oxogalactono-1,4-lactone L-ascorbic acid

Figure 37

the reduction of the C-4 carbonyl group, also
from the opposite face, yielding GDP-l-ga-
lactose (GDP-l-Gal). This reaction is catalysed
by GDP-mannose-3,5-isomerase (EC 5.1.3.18). In
the next step, GDP-l-Gal is probably transformed
to GDP-l-galactose 1-phosphate (l-Gal1P). It is
not yet known if l-Gal1P is an intermediate. It is
shown although this is speculative. l-Gal1P is then
hydrolysed to l-Gal by an unknown enzyme (prob-
ably by sugar-phosphatase, EC 3.1.3.23), and l-Gal
is oxidised to l-galactono-1,4-lactone under the
action of l-galactose dehydrogenase (EC 1.1.1.48).
The final oxidation of l-galactono-1,4-lactone
at C-2 to l-2-oxogalactono-1,4-lactone (l-lyxo-
hex-2-ulosono-1,4-lactone) is catalysed by l-ga-
lactono-1,4-lactone dehydrogenase (EC 1.3.2.3)
using ferricytochrome-c as an electron acceptor.
This lactone then spontaneously isomerises to
l-ascorbic acid (Wheeler et al. 1998; Davey et
al. 1999; Loewus 1999; Smirnoff 2001).
It seems that there exist some other alternative
pathways, revealing a more complex picture of l-as-
corbic acid biosynthesis. For example, GDP-l-gulose
(GDP-l-Gul) and myo-inositol have been suggested
to be another intermediates in l-ascorbic acid bio-
synthesis, indicating that part of the animal path-
way may also be operating in plants (Valpuesta
& Botella 2004).
9.2 l-Ascorbic acid analogues
Microorganisms (including Saccharomyces cere-
visiae yeasts) and higher fungi do not posses the
ability to synthesise l-ascorbic acid from d-al-
doses.18 Instead, they synthesise d-erythro-ascor-
bic acid (d-glycero-pent-2-enono-1,4-lactone), a
five carbon analogue of l-ascorbic acid. This is
thought to perform antioxidant functions in fungi
resembling those performed by l-ascorbic acid in
other eukaryotes. Both compounds have similar

18
However, the synthesis of l-ascorbic acid in e.g. Saccharomyces cerevisiae can be induced by supply of unphysiological
substrates such as l -galactose and various 1,4-lactones, which are converted into l -ascorbic acid via the enzymes
acting in the d -erythro-ascorbic acid biosynthesis.

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Czech J. Food Sci. Vol. 25, No. 3: 101–118

NAD(P) NAD(P)H + H O2 H2O2 CH2 OH

CH2 OH CH2 OH
O O O O O
HO OH HO O HO O O O
HO HO
EC 1.1.1.117 EC 1.1.3.37
OH OH OH OH O OH OH

D-arabinose D-arabinono-1,5-lactone D-arabinono-1,4-lactone D-2-oxoarabinono-1,4-lactone D-erythro-ascorbic acid

Figure 38

redox properties and are substrates for l-ascorbate for counter-ions, and to avoid the mechanistic
oxidase (EC 1.10.3.3). confusion.
The pathway of d-erythro-ascorbic acid biosyn- ACP – acyl carrier protein
thesis shares many features with the pathway for Ado – adenosine
l-ascorbic acid in plants (Loewus 1999; Hancock Ara – arabinose
et al. 2000). The first step, oxidation of d-arabinose ADP – adenosine 5'-diphosphate
(d-Ara), is catalysed by a NAD(P)+-specific d-arab- AMP – adenosine 5'-monophosphate
inose dehydrogenase (EC 1.1.1.117), which oxidises ATP – adenosine 5'-triphosphate
d-Ara at C-1 to produce the corresponding aldo- CoA – coenzyme A as a part of a thioester
nolactone, possibly l-arabinono-1,5-lactone, which DNA – deoxyribonucleic acid
then spontaneously rearranges to the more stable FAD – flavine adenine dinucleotide
d-arabinono-1,4-lactone (Figure 38). The final step, FMN – flavin mononucleotide
the oxidation of d-arabinono-1,4-lactone at C-2, is Fru – fructose
catalysed by an FAD-containing enzyme d-arab- H2PteGlu – 7,8-dihydrofolic acid
inono-1,4-lactone oxidase (EC 1.1.3.37). Finally, the H4PteGlu – (6S)-5,6,7,8-tetrahydropteroylglutamic acid
oxidation product, d-2-oxoarabinono-1,4-lactone Gal – galactose
(d-erythro-pent-2-ulosono-1,4-lactone), spontane- GDP – guanosine-5'-diphosphate
ously isomerises to d-erythro-ascorbic acid. Glc – glucose
Apart from d-erythro-ascorbic acid, other fun- GlcA – glucuronic acid
gal ascorbic acid analogues and their glycosides Gln – l-glutamine
currently known include 6-deoxy-l-ascorbic Glu – l-glutamic acid
acid (a potential source is l-fucose), 6-deoxy- GMP – guanosine-5'-monophosphate
5-O-(α-d-xylopyranosyl)-l-ascorbic acid, 6-de- GTP – guanosine-5'-triphosphate
oxy-5-O-(α-d-glucopyranosyl)-l-ascorbic acid, Gul – gulose
5-O-(α-d-xylopyranosyl)-d-erythro-ascorbic acid, GulA – gulonic acid
5-O-(α-d-glucopyranosyl)-d-erythro-ascorbic acid, Man – mannose
isolated from eatable mushrooms (Basidiomycetes), NADH – nicotinamide adenine dinucleotide
and 5-O-(α-d-galactopyranosyl)-d-erythro-ascorbic NADPH – nicotinamide adenine dinucleotide phosphate
acid isolated from Sclerotinia sclerotiorum (Asco- P – phosphoric acid
myces). Glycosidation (occurring at C-5) of ascorbic PP – diphosphoric acid
acid analogues appears to be a common feature in PPP – triphosphoric acid
fungi. In the presence of H 2O2 and under alkaline SAH – S-adenosyl-l-homocysteine (AdoHcy)
conditions, d-erythro-ascorbic acid is cleaved be- SAM – S-adenosyl-l-methionine (AdoMet)
tween C-2 and C-3 to yield oxalic acid and d-glyceric UDP – uridine-5'-diphosphate
acid (Loewus 1999; Hancock et al. 2000). UMP – uridine-5'-monophosphate
UTP – uridine-5'-triphosphate
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Received for publication February 20, 2006
Accepted after corrections October 30, 2006