Microbial Biology

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Microbial Biology BIOL 3370 Lecture 6
pt iii Vitamins B1, B2 and
biotin (H or B8) April 6, 2009 Opening
Day 2009! Go Cubs Go
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Biotin
Food Sources liver, egg yolk, green
vegetables Functions used in energy and amino
acid metabolism, fat synthesis and
breakdown Deficiency fatigue, loss of
appetite, muscle pain, dry and scaly
skin 
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FYI
Am J Clin Nutr. 2003 Jun77(6)1352-60. Riboflavi
n (vitamin B-2) and health. Powers HJ. Centre
for Human Nutrition, The University of Sheffield,
United Kingdom.
Human dietary info on vitamin B12
http//www.cc.nih.gov/ccc/supplements/vitb12.html
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Thiamin(e) di- (or pyro-)phosphate Phosphorylated
(co-factor) form of thiamin vitamin B1
thiazole
pyrimidine
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Fold of IPDC from Enterobacter cloacae. The
cofactor ThDP and the magnesium ion are included
as ball-and-stick models. The broken line
indicates the disordered loop comprising residues
342355.
thiamindiphosphate-dependent indolepyruvate
decarboxylase
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FUNCTION OF TPP stabilization of acyl
carbanions in biological systems
R can be many, many different things
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Vitamin B1
Some important roles Pyruvate
dehydrogenase Pyruvate decarboxylase Transketola
se Also, a-ketoacid decarboxylase, a-ketoacid
dehydrogenase, acetolactate synthase
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Pentose Phosphate Pathway and Glycolysis are
linked by the B1-dependent enzyme TRANSKETOLASE
(TK) and a transaldolase (ta)
TK
C5 C5 C3 C7 C3 C7 C4 C6 C5
C4 C3 C6
ta
TK
TK moves the two carbon unit
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10 is 5-aminoimidazole ribotide an intermediate
in purine biosynthesis
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Genes for thiamin synthesis
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In 1998 about 3300 tons of Vitamin B1 were made
via chemical synthesis
Why not do it with a non-toxic bacterial strain
that can over-produce the vitamin?
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Vitamin B2 is riboflavin. Riboflavin is
7,8-dimethyl-10-ribityl-isoalloxazine. 7,8-dimeth
yl-10-ribityl-isoalloxazine is
.
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Riboflavin (vitamin B2) is biosynthesized in
plants and in many bacteria. Vegetables and milk
are major sources of the vitamin for
humans. Ruminants can derive vitamin B2 from
their intestinal flora. The daily recommended
allowance for vitamin B2 is 1.8 mg. Although the
flavo-coenzymes are absolutely indispensable in
all cellular organisms, symptoms of riboflavin
deficiency are rarely observed in
humans. However, latent riboflavin deficiency
may be relatively common, especially in women and
adolescents in developing countries .
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e
flavin-adenine dinucleotide
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A riboflavin biosynthesis pathway in bacteria.
Bacillus gene names are underlined.
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GTP cyclohydrolase II
Biosynthesis of riboflavin (9).

• GTP
• 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone
  5'-phosphate
• -
• -
• 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
  5'-phosphate
• 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
• 3,4-dihydroxybutanone 4-phosphate
• 6,7-dimethyl-8-ribityllumazine
• Riboflavin
• ribulose 5-phosphate

lumazine synthase
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Functions as a proton and electron carrier in
metabolic redox chemistry
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Other roles
PPO --protoporphyrinogen IX oxidase a key enzyme
in haem (heme) and chlorophyll
biosynthesis
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Regulation if the rib (riboflavin) operon
Another riboswitch mechanism The nascent message
--the rfn-box-- binds FMN/FAD
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The complexity of commercial over-production of
B2
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Vitamin H or B8 Biotin
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Biotin functions as a CO2 carrier in
carboxylations, decarboxylations, and carboxyl
transfers
Fatty acid synthesis
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Biotinylated proteins are extremely rare in
nature. For example, the only biotin-dependent
carboxylase in Escherichia coli is acetyl-CoA
carboxylase (ACCase EC 6.4.1.2), a multisubunit
enzyme in which one polypeptide is biotinylated
and corresponds to the biotin carboxyl carrier
protein (BCCP). Other bacteria contain one to
three biotinylated proteins. Eukaryotic cells
appear to contain a slightly greater number of
biotinylated proteins. For example, Saccharomyces
cerevisiae contains four or five biotinylated
proteins depending on growth conditions, whereas
mammals are reported to contain four biotinylated
proteins. All these enzymes have crucial
cellular housekeeping functions. More
specifically, ACCase that catalyzes the ATP- and
HCO 3-dependent carboxylation of acetyl-CoA is
recognized as the regulatory enzyme of
lipogenesis methylcrotonoyl-CoA carboxylase
(MCCase EC 6.4.1.4) catalyzes the conversion of
methylcrotonoyl-CoA to methylglutaconyl-CoA, a
key reaction in the Leu degradation pathway
propionyl-CoA carboxylase (PCCase EC 6.4.1.3) is
a key enzyme in the catabolism of odd-numbered
fatty acids and the amino acids Ile, Thr, Met,
and Val and pyruvate carboxylase (PyrCase EC
6.4.1.1) has an anaplerotic role in the formation
of oxaloacetate. The common feature of these
reactions is the transfer of a carboxyl group
from bicarbonate to an acceptor substrate,
utilizing biotin as a carboxyl carrier.
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E. coli BCCP is the 156 amino acyl residue biotin
carboxy carrier protein
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Biosynthesis
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Same thing
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From E. coli
Schematic representation of the biotin regulatory
system. A, The activated enzyme, BirAbio-5'-AMP,
switches between functioning as (i) a biotin
holoenzyme ligase and (ii) a transcriptional
repressor. B, Schematic representation of the
biotin biosynthetic operon and the detailed
sequence of the biotin operon transcriptional
control region bioA through bioF are the genes
that encode the biotin biosynthetic enzymes, O/P
is the transcriptional control region for the
operon, and Pa and Pb are the promoters for
leftward and rightward transcription,
respectively indicated by the arrows. The boxed
region represents the biotin operator sequence.
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BirA is found in many bacteria
Escherichia coli BirA is shown in the first row.
The 2 and 3 helices form the helix-turn-helix
(HTH) structure. The score and the probability of
the candidate HTH motif are given. A score of
lt2.5 is not significant. Non-HTH proteins are
boxed, except BirA from Bacillus cereus, which is
a false-negative prediction