ST JOSEPH’S UNIVERSITY BANGALORE

Name:G VASANTH

REG no.: 21CZBT15

SUBJECT : TERM PAPER

PAPER CODE : TP

Biotin carboxylase and beta-hydroxyacyl thiol Esther dehydrogenase are two different
enzymes involved in distinct metabolic processes. Here's an overview of their functions:

1. Biotin Carboxylase:

Biotin carboxylase is an enzyme that catalyzes the addition of carbon dioxide (CO2) to the
biotin prosthetic group of biotin-dependent carboxylases. This reaction is an essential step
in several important metabolic pathways, including:

a. Fatty acid synthesis: Acetyl-CoA carboxylase, which catalyzes the committed step in
fatty acid synthesis by converting acetyl-CoA to malonyl-CoA, includes biotin carboxylase
as a component.

b. Gluconeogenesis: A crucial stage in the production of glucose from non-carbohydrate

precursors, the conversion of pyruvate to oxaloacetate is catalyzed by the enzyme biotin
carboxylase, which is a component of the pyruvate carboxylase enzyme.

c. Amino acid metabolism: By participating in the branched-chain alpha-keto acid

dehydrogenase complex, biotin carboxylase is involved in the metabolism of a number of
amino acids, including leucine, isoleucine, and valine.

2. Beta-Hydroxyacyl-CoA Dehydrogenase (Beta-HAD): This enzyme is a part of the

catabolic process known as beta-oxidation of fatty acids, which produces acetyl-CoA
molecules that can subsequently enter the citric acid cycle to provide energy. In particular,
beta-HAD catalyzes the third step in the beta-oxidation spiral, which is the oxidation of
beta-hydroxyacyl-CoA to beta-ketoacyl-CoA. Two hydrogen atoms are extracted from the
substrate in this reaction, and the electrons that are left over are sent to the electron
transport chain where they are used to synthesize ATP. As it guarantees the appropriate
development of the beta-oxidation cycle, which culminates in the total oxidation of fatty
acids and the production of energy in the form of ATP, beta-HAD is an essential enzyme in
the breakdown of fatty acids. In conclusion, diverse roles are played by beta-HAD and
biotin carboxylase in various metabolic pathways.

While beta-HAD is involved in the breakdown of fatty acids through the beta-oxidation
pathway, producing acetyl-CoA molecules for energy production, biotin carboxylase is
involved in the addition of CO2 to biotin-dependent carboxylases, which are crucial for
processes like fatty acid synthesis and gluconeogenesis. Four carboxylases are
prosthetically grouped together as biotinylated carboxylases. It is covalently bonded to the
epsilon amino group of a particular lysyl residue close to the active core of active enzymes.
In the placenta, pancreas, kidney, and adipose tissue, acetyl-CoA carboxylase (EC6.4.1.2)
facilitates the elongation of fatty acids; it is absent from the brain. It is found in the cytosol
and mitochondria. An unique isoform of the enzyme is produced in nonlipogenic tissues
(such as the liver, skeletal muscle, and heart) through alternative splicing.

It functions in tandem with beta-oxidation of fatty acids by regulating the activity of

carnitine palmitoyl transferase I. Oxaloacetate), the precursor for glucose production in
gluconeogenic organs (liver, kidneys), is produced by pyruvate carboxylase (EC6.4.1.1). The
only anaplerotic (refilling) process that can restore intermediates in the TCA cycle without
using glutamate or other amino acids is this one. The activity of pyruvate carboxylase rises
as acetyl-CoA concentration rises.

One of the last stages of the catabolism of valine, leucine, isoleucine, methionine,
threonine, and odd-chain fatty acids is catalyzed by protonyl-CoA-carboxylase (EC6.4.1.3)
in mitochondria. Leucine is catabolized by the mitochondrial enzyme 3 methylcrotonyl-
CoA carboxylase (EC6.4.1.4). Protein expression: Research has shown that biotin
increases the availability of messenger RNA (mRNA) for the asialoglycoprotein receptor
(Collins et al., 1988), ornithine transcarbamylase mRNA (Maeda et al., 1996), and hepatic
glucokinase (Romero-Navarro et al., 1999). According to Rodríguez-Meléndez et al. (2001),
low biotin availability lowers pyruvate and propionyl CoA-carboxylase masses and reduces
the expression of biotin-[propionyl-CoA-carboxylase] (EC6.3.4.10).

The interaction with methylated cytosine-binding protein 2 (MeCP2), euchromatic histone-

lysine N-methyltransferase 1 (EHMT1, EC2.1.1.43), and DNA (cytosine-5)-
methyltransferase 1 (DNMT1, EC2.1.1.37) is one of the mechanisms that mediates biotin-
dependent repression of specific genes (Xue and Zempleni, 2013). Other pathways by
which biotin may affect transcription include the biotinylation of nuclear histones H1, H2A,
H3, and H4 by biotinidase, or guanosine 3′,5′-cyclic monophosphate as a second
messenger (Zempleni et al., 2012). The functional importance of this sort of protein
modification is still unknown because biotinylation occurs in just a small fraction of these
histones (<0.1%).

Large multifunctional polypeptides with the biotin carboxylase, biotinyl carboxyl carrier
protein, and carboxyltransferase domains make up animal and fungus ACCs. These
functional elements are found on three distinct polypeptides in prokaryotes (Chapter 3).
The valeryl carboxyl of biotin and the ammonium group on the side chain of a lysine residue
in the biotin carboxyl carrier protein domain form an amide bond that covalently links the
biotinyl moiety. Because of the numerous rotatable bonds that connect the ring structure
to the protein backbone, biotin acts as a swinging arm to influence the transfer of the
carboxyl moiety over the approximately 7 Å distance between the active sites of the two
catalytic domains.

The yeast biotin carboxylase and carboxyltransferase domain crystal structures have been
solved and they most likely correspond to their human counterparts Prokaryotic biotin
carboxylases are active in the dimer form, and isolated biotin carboxylase domains of
eukaryotic ACCs are inactive monomers, indicating that, in the context of the full-length
ACC, they are also likely active as dimers. X-ray crystallography was used to solve the
structure of the biotin carboxylase complexed with soraphen A, a strong polyketide
inhibitor of eukaryotic ACCs. The biotin carboxylase domain, which is composed of three
subdomains—A, B, and C—as well as an AB linker, has a "ATP-grasp" fold Together with the
ATP-binding soraphen, the A and C domains and the AB-linker form a cylindrical shape.

It has been determined that the crystal structures of the yeast biotin carboxylase and
carboxyltransferase domains [2] are similar to those of humans (Fig. 2C and D). Since
isolated biotin carboxylase domains of eukaryotic ACCs are inactive monomers and
prokaryotic biotin carboxylases are active in the dimer form, it is likely that these enzymes
are also active as dimers when found in the context of full-length ACCs. The structure of
the biotin carboxylase complexed with soraphen A, a potent polyketide inhibitor of
eukaryotic ACCs, was solved using X-ray crystallography. The "ATP-grasp" fold is present in
the biotin carboxylase domain, which is made up of three subdomains: A, B, and C,
together with an AB linker (Fig. 2C). The AB-linker, the A and C domains, and the ATP-
binding soraphen form a cylindrical

Each polypeptide in the carboxyltransferase domain is made up of closely related subunits

that are arranged "head-to-tail" when crystallized (Fig. 2D). Two active sites on the dimer
are situated where the opposing subunits' N- and C-terminal domains meet. The primary
chain of residues in the N-terminal domain forms hydrogen bonds with the adenine base,
and the phosphate groups engage with basic residues connected to the N- and C-domains
to bind acetyl-CoA in the active site. Most likely, the carboxybiotin substrate docks with a
brief β-sheet in the C-domain next to acetyl-CoA attached to the other subunit's N-domain.
The outcomes of studies including the mutagenesis of many residues in the active-site
region provide support for the suggested reaction mechanism. Lately

NMR has recently been used to solve the structure of the human ACC2 biotin carboxyl
carrier apoprotein domain (Fig. 2E). This domain's "capped β-sandwich" core structure is
surrounded by lengthy linkers. Similar to its prokaryotic counterparts, the biotinylation site
is situated on a β-hairpin turn.

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Find out more: PMC Notice of Copyright | PMC Disclaimer Protein Sci., 2005 Jun; 14(6):
1570–1580; logo. 10.1110/ps.051373005, PMPMC2253385; PMID: 15930004; CID: The
crystal structure of PfFabZ, a distinct β-hydroxyacyl-ACP dehydratase involved in
Plasmodium falciparum's fatty acid biosynthesis Fritz K. Winkler, Gerd Folkers, Leonardo
Scapozza, Remo Perozzo, and Dirk Kostrewa3. Details about the author Article highlights
Details about licenses and copyrights PMC Notice Visit: Synopsis Plasmodium
falciparum's special β-hydroxyacyl-ACP dehydratase, PfFabZ, is involved in the production
of fatty acids and catalyzes the dehydration of β-hydroxy fatty acids that are attached to
acyl carrier proteins. The arrangement was

Plasmodium falciparum's special β-hydroxyacyl-ACP dehydratase, PfFabZ, is involved in

the production of fatty acids and catalyzes the dehydration of β-hydroxy fatty acids that are
attached to acyl carrier proteins. Using potassium iodide in a quick-soaking experiment,
the structure was solved using single anomalous dispersion (SAD) phasing and refined to
2.1 Å resolution. The crystal structure is the first known structure of a wide substrate-
specific Plasmodium β-hydroxyacyl-ACP dehydratase. A hexamer that manifests as a
trimer of dimers is present in the asymmetric unit. The "hot dog" fold, which is only seen in
a few number of other protein structures, is displayed by each dimer. Equal contributions
from both subunits form each of the dimer's two separate active sites. The majority of the
hydrophobic active site appears like a tunnel bent like an L. The lone hydrophilic site in this
tunnel is formed by the catalytically significant amino acids His 133 and Glu 147′ (from the
opposite subunit) in conjunction with His98′. The flexible, partially visible loop containing
Phe 169's phenyl ring closes the inner end of the active site tunnel.

The genome project discovered a number of extremely intriguing metabolic pathways in P.

falciparum that were previously unknown or hardly defined. Among these metabolic
pathways, a full type-II fatty acid biosynthesis system (FAS-II) could be found (Gardner et
al. 2002). Given the significant structural differences between the cytosolic enzymes of
mammals and yeast and the plastid-associated enzymes found in plants and most
microorganisms, including Plasmodia, the FAS-II pathway presents an especially intriguing
target for therapeutic intervention (Rock and Cronan 1996). In contrast, the latter have a
system known as FAS-II, which is made up of distinct monofunctional enzymes for the
same enzymatic operations, while the former carry a huge homodimeric multifunctional
protein. The fatty acid production of Escherichia coli has been thoroughly investigated, and
it serves

For all living cells, the production of fatty acids is essential. Since bacteria use a type II fatty
acid synthase (FAS II) made up of several different proteins, as opposed to higher species,
FAS II enzymes are a great place to start looking for new antibiotics. An important step in
the FAS II pathway is catalyzed by the enzyme β-hydroxyacyl-ACP dehydratase (FabZ).
Here, we present the crystal and solution structures of Campylobacter jejuni FabZ
(CjFabZ), which exhibit a hexamer with each protomer assuming the distinctive hot dog
shape. We identify the first functioning FAS II enzyme from this pathogen and offer a
foundation for further research on the involvement of FAS II in C. jejuni virulence, in
conjunction with biochemical examination of CjFabZ.

Following sonication-assisted cell lysis, centrifugation at 40,000 g for 20 minutes was used
to extract the entire cell lysate. First, using glutathione-Sepharose 4B beads (GE
Healthcare) and affinity chromatography, the GST-CjFabZ fusion protein was extracted. The
CjFabZ portion was cleaved using 80 units of PreScission protease (GE Healthcare) in 3 ml
of cleavage buffer (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100,
and 1 mM DTT) overnight at 4 °C. The sample was then collected by centrifugation at 1000g
for 5 minutes after the binding step, which lasted for two hours at 4°C, and extensive
washing with PBS in the presence of 0.2% Triton X-100 and 1 mM DTT. Superdex 75 10/300
high resolution gel filtration column was used to mix, concentrate, and further purify the
very pure fractions.

Molecular replacement revealed that the P212121 cell of the crystal's asymmetric unit is
made up of six subunits that together form a hexamer. The final structure is neatly
organized, and all residues' electron densities—aside from the C-terminal residues and the
connecting loop between α3 and β3—were easily interpreted (three to five residues
depending on chains). In the absence of ligands, this loop was also disordered in the FabZ
crystal structures of Plasmodium falciparum and Pseudomonas aeruginosa, indicating a
very flexible area of the structure [13, 14]. The nomenclature of the E. coli β-
hydroxydecanoyl thiol ester dehydratase (FabA) [15] indicates that the monomer structure
of CjFabZ adopts a characteristic mixed β + α 'hot dog' fold (Fig. 2B). The six-stranded,
highly curved anti-parallel β sheet β1/β2/β4/β5/β6/β3 that makes up each subunit is
wrapped around
Even though E. coli is the model organism for the FAS II pathway, questions concerning the
functional assignment of several unidentified homologs from various genome sequences
have been raised by the discovery of distinct gene structures and organizational patterns
among proteobacteria. It was abundantly evident from earlier research that sequence
comparison alone could not be used to infer the biosynthesis routes of fatty acids in
bacteria [5,20]. The results of our analysis indicate that the gene structures encoding C.
jejuni FAS II differ significantly from those of E. coli FAS II. Specifically, the FAS II gene
cluster is absent from the former, and two copies of the genes encoding FabG and FabH
are present; these genes, along with additional C. jejuni FASII enzymes, need to be
functionally annotated. So far, FabZ crystal structures from P. aeruginosa, P. falciparum,
and H. have been solved. The conversion of acetyl-CoA into malonyl-CoA is catalyzed by
ACC (Tong, 2013). The multifunctional protein is composed of carboxyltransferase, biotin
carboxylase, and biotin carboxyl carrier protein. The ACC1 and ACC2 isoforms, sometimes
referred to as ACCα and ACCβ, are expressed in mammals and have a 73% amino acid
sequence identity. The extra 140 amino acid residues in ACC2's N-terminal direct this
isoform toward the outer mitochondrial membrane, whereas ACC1 is cytosolic. Whereas
ACC2 is mostly expressed in tissues with limited lipogenic capacity but high fatty acid
oxidation rates, such as the heart and skeletal muscle, ACC1 is the primary type expressed
in lipogenic tissues where fatty acid production is strong. In these tissues, carnitine
palmitoyltransferase (CPT)-I, which catalyzes the transport of long-chain fatty acids, is
inhibited by malonyl-CoA generated by ACC2.

Mice's lipid buildup in liver and adipose tissues was decreased by specifically inhibiting
Acaca expression in these organs. Conversely, mice with reduced expression of the Acacb
gene (encoding ACC2/ACCβ) exhibit normal lifespan and fertility, elevated fatty acid
oxidation, increased energy expenditure, decreased body fat and weight, improved insulin
sensitivity, and a smaller but functional heart. In rats given a high-fat diet, hepatic steatosis
and hepatic insulin resistance are reversed by antisense oligonucleotides that reduce the
expression of both Acaca and Acacb. Regarding the function of these important enzymes in
controlling fatty acid oxidation, there is some disagreement. Reduced fatty acid oxidation
may be the reason of the elevated hepatic lipid levels in liver-specific double knockout
mice lacking ACC1 and ACC2. There have been hints that

Citrate allosterically activates the activity of both ACC1 and ACC2 in mammals by raising
the Vmax for acetyl-CoA without changing the Km. ACC polymerization and activation are
facilitated by a homodimer of the tiny cytosolic protein MIG12. The MIG12/Spot14
heterodimer-forming Spot14 protein, which is related to MIG12, adversely regulates MIG12
activation. AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA)
catalyze the phosphorylation that inactivates ACC. PKA is triggered by adrenaline and
glucagon, which is mediated by an increase in cellular cAMP concentrations. This is
particularly significant during fasting, when glucagon signaling in the liver predominates
over insulin. An energy-deficient state, such as hunger, exercise, and other conditions,
causes a rise in the AMP/ATP ratio, which activates AMPK, an energy-state sensor. Hepatic
ACC levels are highest while consuming carbohydrates and lowest when fasting or
starving. Insulin activity has been linked to the stimulation of ACC synthesis by
carbohydrates. Low fatty acid synthesis in untreated diabetes mellitus (low insulin) and
restoration of fatty acid production upon insulin treatment highlight the significance of
insulin-mediated regulation of ACC abundance. On the other hand, in obese models with
raised glucose and insulin levels, ACC levels rise and fatty acid production increases.
Fasting and eating cause abrupt changes in ACC quantity, which point to coordinated
transcriptional suppression and activation. There are two distinct genes that encode ACC1
and ACC2. At least three distinct promoters (PI–III) are used to initiate transcription of the
Acaca gene. Acaca messengerRNA (mRNA) is produced through transcription from a PIII
promoter.

Pyridopyrimidine 2 was discovered while screening a sizable pharmacological library

obtained from eukaryotic drug development initiatives. This lead is directed towards the
biotin carboxylase part of ACCase's adenosine-5′-triphosphate (ATP)-binding site. While 2
has nanomolar potency and high selectivity for its bacterial target over a range of
eukaryotic protein kinases, it shares structural similarities with known human targets.
Nevertheless, it is ineffective against significant Gram-positive bacteria and susceptible to
efflux in E. coli [10]. Affinity selection–mass spectrometry library screening produced
benzimidazole hits, which were then refined to imidazo[4,5-b]pyridine 3 using structure-
based drug design. This chemical has an IC50 of 20 nM against the E. coli enzyme, and it is
selective for bacterial biotin carboxylase. Once again, sensitivity to efflux could be a
problem because MICs are only provided

In the process of making wax, malonyl-CoA is initially formed from acetyl-CoA. Acetyl-CoA
carboxylase (ACCase), which has three functional activities—biotin carboxylase (BC),
biotin carboxylate carrier protein (BCCP), and carboxyltransferase (CT)—catalyzes the
process. N-1′ carboxybiotin-BCCP is created when carbon dioxide generated from
bicarbonate is added to a covalently bonded biotin molecule found in the BCCP protein.
Malonyl-CoA is then created by transferring CO2 from acetyl-CoA. BC and CT, respectively,
catalyze these reactions (Figure 1). A flexible arm measuring 1.6 nm in length connects
biotin to BCCP, potentially facilitating its transfer between BC and CT.

Plants have two different forms of ACCase: one is found in plastids and the other in the
cytoplasm. Plasmidal ACCase is found in a heteromeric form in several taxa, including
dicotyledons but not the Poaceae, as dissociable multisubunits of BCCP, BC, and
nonidentical α- and β- subunits of CT. Plasmidic ACCase is found in the Poaceae family in a
multifunctional nondissociable homomeric form, while cytosolic ACCase is homomeric in
all plant species that have been investigated thus far. Malonyl-CoA is supplied by cytosolic
ACCase for the synthesis of polketides and flavonoids as well as for further elongation of
acyl chains, whereas plastidial ACCase provides it for de novo synthesis. Thus, ACCase
plays a crucial part in controlling the synthesis of acyl chains. Thioredoxin reduces the
enzyme in response to light stimulation, activating ACCase. In pea (Pisum sativum), the CT

The first committed step in the production of long-chain fatty acids is catalyzed by acetyl-
CoA carboxylase (ACC) (see Chapter 7.11).36 Two consecutive reactions accelerate the
total reaction (Scheme 3). Using bicarbonate as a CO2 source, the biotin carboxylase
domain first catalyzes the ATP-dependent carboxylation of biotin, which is bound to a
carrier protein. Malonyl-CoA is created in the second step, which involves the transfer of
the carboxyl group from biotin to acetyl-CoA. While a single protein catalyzes both
activities in mammals, two distinct proteins—a carboxytransferase and a biotin
carboxylase—catalyze the activity in bacteria, including Escherichia coli.38 Inhibitors of
the whole ACC reaction are suggested to be beneficial as antiobesity medications in
mammals due to their function in fatty acid production.

The synthesis of malonyl-CoA from acetyl-CoA by acetyl-CoA carboxylase (ACC) is the

initial committed step in FASII. Only a small portion of the entire cellular CoA pool is made
up of malonyl-CoA, which is a product specifically intended for FASII. Log phase E. coli
cells may contain 0.5–1 mM total CoA, of which 25–50 μM is malonyl-CoA. Most Gram-
positive and Gram-negative bacteria have an ACC that is made up of three domains: a
carboxyltransferase (CT), biotin carboxylase (BC), and biotin carboxyl carrier protein
(BCCP) (Figure 3.2). These three domains catalyze two half-reactions that result in malonyl-
CoA: (1) biotin carboxylation on BCCP; and (2) carboxy-biotin transfer to acetyl-CoA to
generate malonyl-CoA. Encoded by the accB gene, BCCP is a homodimer with a molecular
weight of roughly 17 kDa. BC is represented by accg gene BirA, a biotin-apoprotein ligase,
has covalently bound the vitamin biotin to a lysine residue in BCCP. BC uses ATP and
bicarbonate as cofactors to carboxylate the biotin's 1′-nitrogen that is linked to BCCP. In
the process, a temporary intermediate carboxyphosphate is created. Malonyl-CoA is
created in the second half-reaction, which requires CT to transfer the carboxyl group from
BCCP-biotin to acetyl-CoA. Since the protein components separate as a result of cell lysis,
the majority of bacterial ACC multi-domain complexes cannot be isolated. Not every
bacteria adheres to the common ACC structure that has been outlined here. One gene,
expressed by gram-positive actinomycetes, codes for a multi-domain carboxylase protein,
which serves as an ACC. It is composed of two subunits: the beta subunit, which contains
CT, and the alpha subunit, which contains BCCP and BC. Actinomycetes Amphibole

The initial committed step in the production of fatty acids is catalyzed by acetyl-CoA
carboxylase. The entire reaction is made up of two separate half reactions: the transfer of
the carboxyl group from carboxybiotin to acetyl-CoA to create malonyl-CoA is the result of
the ATP-dependent carboxylation of biotin with bicarbonate (Fig. 4). Biotin carboxyl carrier
protein (BCCP), a 16.7 kDa protein, is covalently connected to biotin. For acetyl-CoA
carboxylase to operate, biotin must be connected to BCCP. A particular enzyme called
biotin-apoprotein ligase catalyzes this coupling reaction.

Two distinct protein subcomplexes catalyze the two acetyl-CoA carboxylase half
processes. Biotin carboxylase, a homodimeric enzyme with 55 kDa subunits that is
copurified in a complex with BCCP (also a homodimer), catalyzes the carboxylation of
biotin. Carboxyltransferase is a heterotetramer consisting of two copies of two different
subunits, α and β, that transfers the carboxy group from the biotin moiety of BCCP to
acetyl-CoA. Only the distinct BCCP-biotin carboxylase and carboxytransferase half
reactions are found in cell extracts, while the complete acetyl-CoA carboxylase reaction
(acetyl-CoA to malonyl-CoA) is lost. We assume that the in vivo enzyme consists of a single
copy of every subcomplex, with a total molecular weight of 280 kDa. The four acetyl-CoA
carboxylase subunits' corresponding genes

Escherichia coli Biotin Carboxylase Functions Dependent on the Catalytic Activity of Both
Homodimer Subunits* Janiyani Kamala Bordelon Tee Cronan, Grover L. Waldrop John D. Jr.
Footnotes shown; Open Access DOI: 10.1074/jbc.M104102200 PlumX Metrics One of the
components of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the first
committed step in fatty acid synthesis, is biotin carboxylase, which catalyzes the ATP-
dependent carboxylation of biotin. Enzymatic activity is preserved when the Escherichia
coli biotin carboxylase is easily separated from the other elements of the acetyl-CoA
carboxylase complex. X-ray crystallography was used to identify the three-dimensional
structure of biotin carboxylase. This structure showed that the enzyme is a homodimer
with two active sites, one complete active site per subunit. To comprehend the roles that
each component plays in the overall operation of biotin

Escherichia coli Biotin Carboxylase Functions Dependent on the Catalytic Activity of Both
Homodimer Subunits* Janiyani Kamala Bordelon Tee Cronan, Grover L. Waldrop John D. Jr.
Footnotes shown; Open Access DOI: 10.1074/jbc.M104102200 PlumX Metrics One of the
components of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the first
committed step in fatty acid synthesis, is biotin carboxylase, which catalyzes the ATP-
dependent carboxylation of biotin. Enzymatic activity is preserved when the Escherichia
coli biotin carboxylase is easily separated from the other elements of the acetyl-CoA
carboxylase complex. X-ray crystallography was used to identify the three-dimensional
structure of biotin carboxylase. This structure showed that the enzyme is a homodimer
with two active sites, one complete active site per subunit. To comprehend the roles that
each component plays in the overall operation of biotin

The initial committed and rate-limiting step in the production of fatty acids is catalyzed by
Escherichia coli acetyl-CoA carboxylase (1). According to the arrangement in Fig. 1, the
overall reaction catalyzed by acetyl-CoA carboxylase progresses via two half-reactions.
Three different components are needed for acetyl-CoA carboxylase to carry out this two-
step reaction: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase.
The first half-reaction in Figure 1 is an ATP-dependent carboxylation of the vitamin biotin,
which is catalyzed by the biotin carboxylase component. As shown in Fig. 1, the biotin
carboxyl carrier protein, or BCCP, is covalently bound to biotin in vivo. The
carboxyltransferase component catalyzes the second half-reaction, which involves the
transfer of the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA. All
three of the components of acetyl-CoA carboxylase are found on one

For a number of reasons, the E. coli biotin carboxylase component encoded by the accC
gene has emerged as the paradigmatic system for investigating biotin-dependent
carboxylation processes. First, the enzyme's gene has been overexpressed, sequenced,
and cloned (4, 5). Second, the only three-dimensional model of a biotin-dependent
carboxylase that has been determined is the enzyme's crystal structure (6, 7). The
structural analysis of biotin carboxylase verified that, unlike other multisubunit enzymes
like aspartate transcarbamylase (8) and ribulose-1,5-bisphosphate carboxylase (9), the
enzyme is a homodimer with two well-separated active sites devoid of residues from both
subunits. This phenomenon is known as a shared active site. Rather, every biotin
carboxylase subunit has its entire active site. This begs the inquiry, "Why does

Novagen supplied the His-Bind resin, which was utilized to purify proteins with a poly-
histidine tag. FLAG octapeptide (NH2-DYKDDDDK-COOH) was produced at the University
of Illinois' protein synthesis laboratory. It was a Sigma anti-FLAG M2 affinity gel. Proteins
were concentrated using colloidal membranes from Schleicher and Schuell.Sigma
provided the adenosine 5′-triphosphate (ATP), NADH, and lactate dehydrogenase.Roche
Molecular Biochemicals supplied the pyruvate kinase.[1–14C]Acetic acid (sodium salt) was
obtained from American Radiolabeled Chemicals (St. Louis, MO) and had a specific
activity of 56.7 mCi/mmol.T4 DNA Ligase and all restriction endonucleases were obtained
from New England Biolabs.Qiagen provided the agarose gel extraction and plasmid DNA
isolation kits.The remaining reagents were acquired from Sigma.Strains of Bacteria and
Media BLR(DE3) pLysS

Plasmid pGLW1 (Table I) encoding wild-type biotin carboxylase was cut with EcoRI and
BamHI. The 1.35-kilobase insert was then ligated to pMTL23, which was cut with the same
enzymes to yield plasmid pKJ1, which had a NcoI site upstream of the accC coding
sequence. It should be noted that we will refer to the gene (and encoded protein) encoding
a wild-type active site as wild type, even though the gene is not strictly wild type due to the
added sequences. After being cut with NcoI and NdeI, this plasmid was ligated to the FLAG
oligonucleotide to produce pKJ2. pKJ2 was cut with NcoI-BamHI and ligated to pET14b,
which was also cut with the same enzymes, to produce plasmid pKJ3, which is the FLAG-
tagged gene from the T7 promoter. To verify that the sequence was there, the plasmid insert
was sequenced.

Using spectrophotometry, the biotin carboxylase activity was ascertained. By monitoring

the oxidation of NADH at 340 nm and utilizing pyruvate kinase and lactate dehydrogenase
to detect the formation of ADP, the rate of ATP hydrolysis was determined. 17.5 units of
lactate dehydrogenase, 10.5 units of pyruvate kinase, 0.5 mm of phosphoenolpyruvate, 0.2
mm of NADH, 8 mm of MgCl2, 15 mm of potassium bicarbonate, and 100 mm of HEPES at
pH 8.0 were present in each reaction. A quartz cuvette with a route length of one
centimeter was used for all reactions, with a total capacity of 0.5 ml. ATP was introduced to
the reaction and kept constant at 3.0 mm until the biotin kinetic parameters were
established. Because biotin has a high K m, KCl had to be added to the process in order
increase.

Three distinct kinds of biotin carboxylase dimers produced in vivo (Fig.3) when the genes
for differentially tagged biotin carboxylase were co-expressed from a plasmid (Fig. 2). There
were two His-tagged subunits (named HH) in one sort of dimer. Another dimer type had
both of its subunits FLAG-tagged (FF), while the third dimer had one subunit His-tagged
and the other FLAG-tagged (HF). Small amounts of a dimer comprising a chromosomally
encoded wild-type (WT) untagged subunit plus either a His- or FLAG-tagged subunit may
also form. First, an immobilized nickel affinity column was used to elute the crude E. coli-
soluble extract, which bound all His tag-containing biotin carboxylase dimers (HH and HF)
and removed the FF dimer.

Escherichia coli Biotin Carboxylase Functions Dependent on the Catalytic Activity of Both
Homodimer Subunits* Janiyani Kamala Bordelon Tee Cronan, Grover L. Waldrop John D. Jr.
Footnotes shown; Open Access DOI: 10.1074/jbc.M104102200 PlumX Metrics One of the
components of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the first
committed step in fatty acid synthesis, is biotin carboxylase, which catalyzes the ATP-
dependent carboxylation of biotin. Enzymatic activity is preserved when the Escherichia
coli biotin carboxylase is easily separated from the other elements of the acetyl-CoA
carboxylase complex. X-ray crystallography was used to identify the three-dimensional
structure of biotin carboxylase. This structure showed that the enzyme is a homodimer
with two active sites, one complete active site per subunit. To comprehend the roles that
each component plays in the overall operation of biotin

After being transformed with the medium copy expression vector pBAD30, which contains
accC genes encoding either the wild type or one of the four mutant biotin carboxylases,
derivatives of the E. coli wild-type strain MG6155 were cultivated until early log phase was
attained. After 40 minutes of adding arabinose to promote gene expression, 10 minutes
were spent adding [1-14C]acetate. After adding trichloroacetic acid to halt the labeling
reaction, the lipids were removed, and scintillation counting was used to evaluate whether
radioactivity had been incorporated. The information is calibrated to a 600 nm culture
turbidity of 1.0 A.

Refernce : Structure and function of biotin-dependent carboxylases

Analysis of the Reaction Mechanism of Biotin Carboxylase

1. Yuko Ito

2. ,
3. Hiroki Kondo Theoretical

4. ,
5. Yoshihito Shiota

6. , and
7. Kazunari Yoshizawa