PEROXISOME METABOLISM

Presented by : Rabina Ramtel

M.Sc. Clinical Biochemistry
OUTLINES:
 Introduction

 Peroxisome biogenesis

 Function of peroxisomes

 Disorder associated with peroxisomes

 It is a microbody of about 0.3-1.5 micrometer in
diameter
 Have a lipid bilayer membrane

 Consists of crystalline core with peroxins

 Peroxisomes are called so; because they produce
hydrogen peroxide (H2O2)

 Also known as the organelle behind the film

“Lorenzo’s oil”

 Consistsof enzymes such as catalase, D-amino acid

oxidase, uric acid oxidase, alpha- hydroxy acid
oxidase etc

 Enzymatic marker for peroxisome is catalase

 Peroxisomes can be isolated by differential
centrifugation

 Differential centrifugation results in a rough

fractionation of the cytoplasmic contents

 Furtherit is purified by isopycnic (“same density”)

centrifugation.

 Thetissue homogenate is first centrifuged at low-

speed(1,000 g, 10 min)

 Supernatant then is subjected to medium-speed

centrifugation (20,000 g, 20 min)
 Results
in pellets containing mitochondria, lysosomes
and peroxisomes

 The pellet, which contained peroxisomes and

mitochondria, was resuspended in MS buffer (0.65 M
sorbitol, 5 mM MES, pH 5.5) and was placed on top
of a gradient of Nycodenz (17%, 25%, 35%, 50%) in
MS buffer.

 Aftercentrifugation at 116,000 g for 2h, peroxisomes

are present in fractions 2 to 8.
 The pellet from the aforementioned 20,000 g
centrifugation was resuspended in 10 volumes of Ti8
buffer (Tris 10mM, pH 8.0 and PINS (1 mM EDTA, 0.2
mM PMSF, 2 μg leupeptin/ml, 2 μg aprotinin/ml, and 0.4
μg pepstatin A/ml))

 separated at 200,000 g for 1 h.

 The pellet, with the peroxisomal membranes, was

resuspended again in Ti8 buffer

 By addition of 0.1 M sodium carbonate and subsequent

centrifugation at 200,000 g for 1 h, the peroxisomal
membranes were separated from the proteins which were
associated with but not integral to the membranes.
 Peroxisomes proliferate by growth and division of
pre-existing peroxisomes or could arise de novo.

 Its biogenesis is unique as it lack their own DNA

 Steps :
 formation of the peroxisomal membrane
 Import of proteins into the peroxisomal matrix
 Proliferations of the organelles
Peroxisome Type of PTS PTS* amino Role in
matrix protein acid peroxisome
sequence metabolism

Acyl-CoA oxidase 1 –SKL Fatty acid

I metabolism
Alanine 1 –KKL Glyoxylate
glyoxylate metabolism
aminotransferase

Alkyldihydroxyac 2 – Plasmalogen
etonephosphate RLVLSGHL– synthesis
synthase

Catalase 1 –KANL H2O2 metabolism

D-bifunctional 1 –AKL Fatty acid
protein metabolism
 Lipid and anaplerotic metabolism

 Production of hydrogen peroxide

 Reduction of reactive oxygen species (ROS)

 Biosynthesis of plasmalogens

 Detoxification of alcohol in liver cells

 Remove amine group from amino acids and convert

it to ammonia prior to excretion
 In
the liver, peroxisomes are also involved in the
synthesis of bile acids, which are derived from
cholesterol.
Lipid and anaplerotic metabolism
One of the major functions of peroxisomes concerns
their role in lipid metabolism, which includes:

 fatty acid beta-oxidation

 fatty acid alpha-oxidation

 ether phospholipid synthesis

Beta-Oxidation
 Occurs when fatty acids chains are too long to be
handled by mitochondria

 Peroxisomal beta-oxidation does not degrade fatty

acids completely

 acts as a chain-shortening system, catalyzing only a

limited number of beta-oxidation cycles.

 The fatty acids are activated to their acyl CoA

derivatives at the peroxisomal membrane and the
beta- oxidation occurs at the peroxisomal matrix
MODELS FOR THE IMPORT OF FATTY ACIDS INTO
THE PEROXISOMES
Alpha(α)-oxidation
 Alpha oxidation occurs in those fatty acids that
have a methyl group(CH3) at the beta-carbon, which
blocks beta oxidation.

 It removes one of the carbon unit adjacent to the α

carbon from the carboxylic end in the form of CO2

 Phytanic acid acts as the substrate

 peroxisomes is the cellular site

 No production of ATP
Biosynthesis of plasmalogens

• Plasmalogens (PLs) were first described by Feulgen

and Voit in 1924

• A family of phospholipids in which one of the

hydrocarbon chains is joined to glycerol by an ether
bond rather than an ester bond

• Important membrane components in heart and brain.

• In human heart tissue, nearly 30–40% of choline
glycerophospholipids are plasmalogens.

• 32% of the glycerophospholipids in the adult human

heart , 20% in brain and up to 70% of myelin sheath
ethanolamine glycerophospholipids are plasmalogen

• It’s Biosynthesis begins with association

of peroxisomal matrix enzymes GNPAT (glycerone
phosphate acyl transferase) and AGPS (alkyl-
glycerone phosphate synthase) on the luminal side of
the peroxisomal membrane
 Impairedplasmalogen biosynthesis also leads to
Peroxisome biogenesis disorders.

 In these cases, the peroxisomal enzyme GNPAT,

necessary for the initial steps of plasmalogen
biosynthesis, is mislocalized to the cytoplasm where
it is inactive.

 Genetic mutations in the GNPAT or AGPS genes can

result in plasmalogen deficiencies, which lead to the
development of rhizomelic chondrodysplasia
punctata (RCDP) type 2 or 3, respectively
Ethanol metabolism

 Oxidation of ethanol can also occur in peroxisomes

via the activity of catalase.

 However, this oxidation pathway requires the

presence of a hydrogen peroxide (H2O2) generating
system and as such plays no major role in alcohol
metabolism under normal physiological conditions
Production of H2O2
Role of peroxisome in bile acids synthesis

 In the early 1980s, the first clues were obtained

indicating the importance of peroxisomes in the
biosynthesis of bile acids.

 Peroxisomes play an important role in the

biosynthesis of bile acids as peroxisomal beta-
oxidation step is required for the formation of the
mature C24-bile acids from C27-bile acid
intermediates.

 Inaddition, de novo synthesized bile acids are

conjugated within the peroxisome.
 The primary bile acids, cholic acid (CA) and
chenodeoxycholic acid (CDCA), are formed from
cholesterol

 Its biosynthesis occurs by two pathways:

 Classical pathway

 Alternate pathway
 Steps involved in bile acid biosynthesis are:

 Modification of ring structure of cholesterol (steroid

nucleus)

 Oxidation of the sterol side chain

 Cleavage of the side chain

 Conjugation with an amino acid, either taurine or

glycine.
PEROXISOMAL STEPS IN BILE ACID BIOSYNTHESIS
-METHYLACYL-COA RACEMASE (AMACR)
BRANCHED-CHAIN ACYL-COA OXIDASE (BCOX)
STEROL CARRIER PROTEIN X (SCPX)
BILE ACYL COA AMINO ACID N-ACYL TRANSFERASE (BAAT)
Role of peroxisome to remove amine group
from amino acids and convert it to ammonia
prior to excretion

 Removal of the α-amino group is the first step

in catabolism of amino acids

 accomplished oxidatively or nonoxidatively

 Oxidative deamination is stereospecific and is

catalyzed by L- or D-amino acid oxidase.
 Impaired peroxisomal function results in a number of
multisystem diseases and are grouped as:

 Group 1: Peroxisomal Biogenesis Disorders (PBD)

 Group 2: Single Peroxisomal Enzyme Defects

involving β-Oxidation. Peroxisomes are
morphologically intact but their function is defective

 Group 3: Single Peroxisomal Enzyme Defects

Without β-Oxidation Involvement
 Refers to a group of related conditions that have
overlapping signs and symptoms and affect many
parts of the body.

 The spectrum includes:

 Zellweger syndrome (ZS), the most severe form

 Neonatal adrenoleukodystrophy (NALD), an

intermediate form

 Infantile Refsum disease (IRD), the least severe

form.
 Recently,Heimler syndrome was recognized as a
peroxisome biogenesis disorder within the
Zellweger spectrum and added to the (very) mild
end of the clinical spectrum.

 Caused by mutations in genes that encode

peroxins, proteins required for the normal
assembly of peroxisomes.

 Cultured primarily skin fibroblasts obtained

from patients shows impaired fatty acid beta-
oxidation, phytanic acid alpha-
oxidation, pristanic acid alpha-oxidation, and
plasmalogen biosynthesis.
ZELLWEGER’S SYNDROME
 ZellwegerSyndrome was discovered by an Switz-
American pediatrician Hans Zellweger.

 Also called as Cerebrohepatorenal syndrome; CHR

 Congenital peroxisome biogenesis disorders

 Caused by mutations in genes that encode peroxins

 Enzymes produced in the cytoplasm are unable to cross

the membrane barrier and enter the matrix of the
peroxisomes.
 Characterized by presence of ghost peroxisomes in
the cells of an individual

 This
will result in failure to break down lipids, and
cannot contribute to the production of Myelin.

 In1978, Hansen et al. were the first to report a

defect in bile acid synthesis in Zellweger syndrome

 Accumulation of C27-bile acid intermediates showed

that Zellweger patients were not able to cleave the
side-chain of these precursors and thus could not
form mature C24-bile acids.
 Also accumulate VLCFAs, pristanic acid, phytanic
acid, pipecolic acid in plasma and have a deficiency
of plasmalogens in erythrocytes.

 The
incidence of ZSDs is estimated to be 1 in 50,000
newborns in the United States
Refsum’s Disease

 Adult Refsum’s Disease

 Named after Norwegian neurologist Sigvald
Bernhard Refsum

 Autosomal recessive peroxisomal disorder

 Caused by the impaired alpha-oxidation of branched

chain fatty acids resulting in buildup of phytanic
acid and its derivatives in the plasma and tissues.

 Defect in enzyme phytanoyl CoA hydroxylase

(Phytanic acid oxidase)
 Phytanic acid is acumulated in brain and other
tissue

 Adult Refsum disease may be divided into subtypes:

 Adult Refsum disease 1

 Adult Refsum disease 2.

 The former stems from mutations in the phytanoyl-

CoA hydroxylase (PAHX or PHYH) gene, on the
PHYH locus at 10p13 on chromosome 6q22-24.
 Refsum disease 2 stems from mutations in the
peroxin 7 (PEX7) gene.

 This mutation on the PEX7 gene is also on

chromosome 6q22-24, and was found in patients
presenting with accumulation of phytanic acid with
no PHYH mutation.

 Lab Findings includes:

 Plasma Level of phytanic acid > 200µmol/L
 Normal< 3oµmol/L
 Infantile Refsum’s Disease
 A peroxisome biogenesis disorder resulting from
deficiencies in the catabolism of very long chain fatty
acids and branched chain fatty acids (such
as phytanic acid) and plasmalogen biosynthesis.

 Lab findings:
1. Phytanic acid in the serum is more than 30µmol/L
and less than 200µmol/L

2. VLCFA and LCFA in serum is increased

 Cardinal features of refsum disease are:
 Retinitis pigmentosa

 Chronic polyneuropathy

 Cerebellar ataxia

 Elevated levels of proteins in csf

 Skeletal malformations
 Molecular Toxicology of Refsum’s Disease

 PA is directly toxic to ciliary ganglion cells and

induces calcium –driven apoptosis in purkinji cells

 Recent studies has found that PA has a Rotenone

like action in inhibiting complex –I and producing
reactive oxygen species

 Hence neuronal cells and retina are prime tissue

affected in Refsum’s disease
Adrenoleukodystrophy (ALD)
 Can be classified as X-linked adrenoleukodystrophy
and neonatal adrenoleukodystrophy

 disorder of peroxisomal fatty acid beta

oxidation which results in the accumulation of very
long chain fatty acids in tissues throughout the body.

 A rare, genetic disorder characterized by the

breakdown or loss of myelin

 most severely affected tissues are the myelin in

the central nervous system, the adrenal cortex, and
the Leydig cells in the testes.
 Adrenoleukodystrophy has an estimated incidence
of around 1 in 20,000–50,000

 ALD is caused by mutations in ABCD1, a gene

located on the X chromosome that codes for ALD, a
peroxisomal membrane transporter protein.

 Forms of X-linked ALD include:

 Childhood-onset ALD

 Addison's disease

 Adrenomyeloneuropathy
 Alpha-methylacyl-CoA racemase (AMACR)
deficiency
 This enzyme (encoded by AMACR) plays a key role in the
breakdown of pristanic acid and the C27-bile acid
intermediates di- and trihydroxycholestanoic acid.

 As a consequence of the impaired degradation of pristanic

acid, both pristanic acid and phytanic acid accumulate
with pristanic concentrations much more elevated than
phytanic acid concentrations

 AMACR deficiency and classic Refsum disease can be

distinguished by screening peroxisome metabolites in the
plasma, followed by fibroblast studies and molecular
genetic testing.
Rhizomelic chondrodysplasia punctata type1
(RCDP1)
 caused by pathogenic variants in PEX7

 RCDP is associated with three characteristic

abnormalities:
 deficient ether lipid synthesis
 deficient phytanic acid oxidation
 failure to process peroxisomal β-ketothiolase to its
mature form

 level of phytanic acid elevated

 Wanders RJ, van Grunsven EG, Jansen GALipid
metabolism in peroxisomes: enzymology, functions
and dysfunctions of the fatty acid alpha- and beta-
oxidation systems in humans.

 Sacha Ferdinandusse; Sander M.Houten.

Peroxisomes and bile acid biosynthesis
https://doi.org/10.1016/j.bbamcr.2006.09.00

 Ronald J. A. Wanders. Et.al. Bile acids: the role of

peroxisomes
 Berger, J.; Gärtner, J. (2006). "X-linked
adrenoleukodystrophy: Clinical, biochemical and
pathogenetic aspects". Biochimica et Biophysica
Acta (BBA) - Molecular Cell Research.

 Steinberg,
S.; Dodt, G.; Raymond, G.; Braverman,
N.; Moser, A.; Moser, H. (2006). "Peroxisome
biogenesis disorders". Biochimica et Biophysica
Acta (BBA) - Molecular Cell Research

 Wouter F. Visser et.al. Metabolite transport across

the peroxisomal membrane
 Lehninger-Principles of Biochemistry 5th edition

 De Duve C (Apr 1969). "The peroxisome: a new

cytoplasmic organelle". Proceedings of the Royal
Society of London. Series B, Biological Sciences.
173(1030):71-83.
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