NUTRIENT ACQUISITION : DIGESTION & TRANSPORT

DISACCHARIDES

Examples: Sucrose, maltose and cellobiose

May be transported into fungus: Intact or Hydrolyzed before being transported.

E.g., In S. cerevisiae, sucrose was converted to glucose and fructose at the cell surface and the monosaccharides were transported.

• Isolated cell walls of S. cerevisiae contained most of the invertase.

• Invertase was solubilized by treated with snail digestive enzyme in mannitol.

• Protoplast were unable to ferment sucrose, but could ferment glucose

• Maltose was not located in wall, but retained in the protoplast.

• Yeast cannot ferment maltose unless the have been grown on maltose.

CELLULOSE

Structure : microfibrillar substance of linear molecules

packed into crystalline arrays interspersed with

amorphous regions.

•Non- ordered structure•Helps in β-linkage to adapt to microenvironment

Native cellulose : Insoluble : Comminuted to produce fine particulate suspension

Modified, soluble cellulose derivatives

Carboxymethyl cellulose ( CMC) and Hydroxyethyl cellulose ( HEC )

- Thickener in Food

Umbelliferyl cellodextrins• Chromogenic substances• Enzyme activities measured based on the

colored products

Cellobiohydrolase Endoglucanases

Digest

ONLY Amorphous Region Crystalline Arrays and Amorphous Region

• CBHI and EGI have greater than 50% nucleotide sequence similarity and about 45% amino acid sequence similarity.

• CBHII and EGIII were unrelated to each other or the first pair.

Two reasons for the expression of the genes for the enzymes in Saccharomyces cerevisiae:

• Since S. cerevisiae has no known exocellular cellulases, expression of the genes individually resulted in single-enzyme activities with no cross contamination.

• Since cellulose substrates are highly variable, conversion of cellulose to glucose may not be optimal with the native mix from Trichoderma reesei.

• Expression of the four cloned cellulase genes of T. ressei in S. cerevisiae succeeded by using cDNA clones from the mRNAs to eliminate the introns that were not correctly spliced by yeast, and by providing suitable yeast promoters.

• The recombinant enzymes from S. cerevisiae were active toward the natural substrates, barley β-glucan and lichenin, and several artificial substrates.

• The specific activity and binding of the recombinant CBHII were reduced in comparison with the natural enzyme, suggesting that the hyperglycosylation affected activity.

GROUP 2

STARCHES AND HEMICELLULOSE

β-glucans have β-1,3 and β-1,6 linkages Starches and glycogens have α-1,4 and

α-1,6 linkages Produced by fungi, algae, vascular

plants, animals Sources of carbon and energy Fungal walls usually contain β-1,3 and

β-1,6 glucans (β-glucans) which are associated with wall fractions or the periplasmic space

Many glucanases have a nutritional role

The hydrolysis of α-1,4 linkages of starch and glycogen is catalyzed by α- and β-amylases and glucoamylases

Amylases are endoglucanases-yield maltose and glucose, also limit-dextrins with α-1,6-branching(debranching enzyme required)

Glucoamylases are exoglucanases-hydrolyze glucose

Many fungi produce amylases and glucoamylases which cleave α-1,4-bonds to yield glucose

Fungi lacking glucoamylase require maltase

Hemicellulose – composed of mixtures of polysaccharides that have different sugar monomers-glucose, galactose, mannose, arabinose, xylose, glucuronic acid

These are alkali-soluble extractable polysaccharides of plant cell walls

Mostly heteropolymeric-have 2 or more sugar monomers, linear or branched, and acetylated

Enzymes required to utilize these materials-xylanase, debranching xylanase, mannanase, galactanase, arabinosidase, glucuronidase, acetly esterase

LIGNIN COMETABOLISM• Lignin is the most abundant carbon

in aromatic form.• Lignin is a branched polymer of

cinnmyl olcohol-derived monomers.• Lignin impregnating material in wood

which protects polysaccharides component from enymatic attack.

Aspects of lignin decompositions: Fungi are the only organisms that

extensively degrade lignin to C02 . Degradation occurs by

predominantly oxidative without releasing its monomer into solution.

Does not provide primary source of carbon and energy for fungal growth.

Decay fungi are grouped into three principal types according to form decay;1)White rot

Basidiomycetes and few Ascomycetes. Capable of complete mineralization of lignin.

2)Brown rot Basidiomycetes Degrade cellulose and hemicellulose

preferentially with limited degradation of lignin.

3)Soft rot Ascomycetes and Deuteromycetes. Various lignolytic abilities on lignin. ( either

no, little or great atttack).

The requirement for primary source of carbon energy for degradation of another compund is known as cometabolism.

Addition of glutamate, glutamine or histidine suppressed ligninase activity.

Exoenzymes involve in lignin degradation:

1)Lignin peroxidase and Manganese (Mn) peroxidase.

Contain Fe-heme prosthetic groups. Coooperative action of these leads to

exocellular “combustion” that depolymerizes lignin.

2)Laccase Cu-containing exoocellular benzenediol

oxidase and other phenol oxidase. May acts synergistically with other enzymes. Able to generate H2O2.

PROTEIN Source of:

Nitrogen Sulphur

Large size polymer Digested wt exocellular protease into amino

acid/peptides Protease regulation through:

Induction(presence of protein) Catabolite repression(when protein appear

as sole C/N/S source )

Protease classification: Exohydrolases : cleave peptide terminally

Aminopeptidase from N terminus Carboxypeptidase from C terminus

Endohydrolases: cleave peptide internally Together endohydrolases create short

peptide substrates availability for exohydrolases rapid protein digestion

Source: http://maptest.rutgers.edu/drupal/?q=node/27

Source: http://www.uwplatt.edu/~sundin/354-7/l547-46.htmSource: http://upload.wikimedia.org/wikipedia/commons/9/93/Tetrapeptide_%26_Aminopeptidase_V.1.svg

EXOENZYME OF PHYTOPATHOGENS

Exocellular enzymes contribute in fungi pathogenic process.

Enzyme activities digest insoluble host materials that are then taken up by the fungus for growth.

Examples:- Cutinases- Pectinases- Hemicellulases, cellulases and lipases

CUTINASES

Are esterases. Penetrate waxes, cutin, and suberin

protective barrier on outer surface of plants.

Inducible by cutin monomers.

PECTINASES Complex polysaccharide composed of -1,4-

galacturonic acid with rhamnose side chains. Attack pectic material of the middle lamellae

and primary walls of plant cells which are responsible for cementing the host cells and wall components together.

Enzyme activites:I. Release methyl groups from carboxyls(Pectin

methyl esterase).II. Hydrolyze -1,4-galacturonosyl bonds

(Hydrolases).III. Lyse -1,4-galacturonosyl bonds by 𝛼

rearrangement of the hydrogens ( Transeliminases or lyases).

HEMICELLULASES, CELLULASES, AND LIPASE Hemicellulases consist of galactanases,

arabanases, xylanases, and mannanases. Cellulases : Enzyme activity:I. Cleave cellobiose unit from non reducing ends of

cellulose ( Cellobiohydrolase).II. Hydrolyze internal bond of amorphous cellulose,

cellodextrins and cellulose derivatives ( Endoglucanase).

III. Hydrolyze cellobiose and oligocellodextrins to glucose ( Cellobiase)

- Exhibit multiple forms.- Glycoprotein.- Very resistant to denaturation.

Lipases : Attack plasmalemma. Lipases A1 and A2 acted on

diacylphospholipid. Lysophospholipases L1 and L2

deacylate monoacyl product of A enzyme.

Phospholipase B cleaves both fatty acids, but it may be a mixture of A and L lipases.

EXOENZYMES OF ZOOPATHOGENS

Enzymes: Proteases:

leucine aminopeptidase elastase collagenase carboxypeptidases

Lipases Acid and alkaline phosphatases Plasmacoagulase Neuraminidase

Production: In vivo Function: Attack and digest host Toxin which consist of ezymes

Eg: canditoxin of Candida albicans

Correlation of enzyme to fungal pathogenicity Eg: phospholipase reponsible

for C. albican pathogenicity However phospholipase activity

repressible by presence of glucose, sucrose, galactose

• Transport mechanism • Energy coupling in active

transport

Transport

GROUP 4LEE PEI ZHEN

ZATUL NAJIHAHNURULAIN JAMARI

NURUL HAFIZAHSITI ROSLINA

MUHAMMAD FAKHRULAZRIFARAH HANIM

TRANSPORT MECHANISM Basic mechanism for communication of cells. Plasmalemma: semipermeable layer of hyphal

surface that regulate flow of material. 2 types of mechanism: passive and active transport Passive transport : movement of substances affect

by gradient concentration. Active transport : substances moves against

concentration gradient which required energy(ATP). Substrate accumulate at higher concentration on

one side of membrane. Accumulation inside hyphae can occur through other

than active transport.

Insoluble sink, immobile binding sites or metabolic sinks may reduce the transport activity( effective concentration) causing net directional movement.

Example: plant cell synthesis oxalic acid causing crystallization of calcium oxalate resulting high accumulation of calcium in cells.

This movement well described in Donnan equilibrium involves formation of immobile ions.

Rapid change such as phosphorylation is the mechanism for accumulation occur in passive transport.

Most substances that move across membrane required help of carrier molecule.There are 3 carrier theory that has been developed.

1. Without carrier molecule, the rate of transport to increase proportionally to

increased external concentration.

2. Carrier-mediated transport are inhibited with some degree of

specificity by substance active in low concentration

3.Similar substances often competitively inhibit transport of the

substrate

Countertransport of these substrate may occur when they are previously loaded into hyphae.

Countertransport is the outward movement of compund in response to the inward movement of substance.

ENERGY COUPLING IN ACTIVE TRANSPORT

Energy coupling in active transport are partially understood.

However, coupling of ATP hydrolysis to proton transport is generally agreed in which it generates driving force for solute transport [47].

2 types of energy coupling which are direct and indirect

DIRECT- COUPLING MODELS Energy of ATP hydrolysis is utilized in the

movement of binding proteins or in the dissociation of substrate.

As ATP-dependent conversion of substrate occur, metabolically driven transport occurs by maintain of low internal concentration of substrate.

This is the passive diffusion that behaves like directly coupled active transport since these mechanism required ATP synthesis.

INDIRECT- COUPLING MODELS These models based on Mitchell hypothesis.

These hypothesis stated that, motive force involves an ATP-generated proton gradient across membrane.

It can be accomplished by a substrate-carrier-proton complex diffusing across the membrane or by molecular gate.

Molecular gate involves protein conformation changes, coupled with proton movement between outer and inner faces of plasmalemma.

GROUP 5

MEMBRANE POTENTIALS AND PH CHANGES

MEMBRANE POTENTIALS

Means the differences in charge across the plasma membrane.

Can be affected by: Transportable substrate (potassium,

glucose, or amino acid) caused transient depolorization

Metabolic inhibitors (cyanide)

PH CHANGES

Attributed to the operation of proton pump. External pH acidic (pH 6 or less). Internal pH (6.5 to 6.8)

provide proton gradient. Transport system of fungi inhibited pH > 7. Affected by

Uncoupling inhibitors (eg. Azide) Transportable substrate

Fungal hyphae grow measured by vibrating microelectrode.

ELECTROGENIC MEMBRANE ATPASES

MEMBRANE ASSOCIATED ATPASES

Different: In cellular locations Ion translocating abilities Organisms that have been found

3 types found in fungi H +- ATPases located in plasmalemma H +- ATPases on vacuole H +- ATPases of mitochondrion

TRANSPORT FUNCTIONS

ATP SYNTHASE FUNCTION

Plasmalemma H +- ATPases

A single monomeric peptide of 100 kDa Very abundant (20-40%) Forms an outwardly directed H+ pump (membrane

potential)

Vacuolar ATPase Translocated protons out of the cytoplasm but into

vacuolar contents Heteromultimeric protein similar to the mitochondrial

ATPase

Both coupled ATP hydrolysis to proton translocation > pH gradients

Both involved in transportation

ROLE OF PLASMALEMMA ATPASE

Developed through the use of inhibitors and isolated ATPase in artificial membrane vesicles.

Ionophores Collapse the membrane potential

simultaneously with collapse of H+/ion gradients.

Stimulated ATPase, inhibit transport

Inhibitors of ATPase function Several types Differed in- vivo and in- vitro experiments

ATPASE MUTANTS Selected in S. cerevisiae and

Schizosaccharomyces pombe Resistance to several drugs

Mutants with reduced H+-ATPase activity Proton efflux activity decreases Growth rates reduce More acidic intracellular Ph Cell proliferation increases

Conclusions:ATPases play important role in proton pumping and intracellular pH regulation

CATION TRANSPORT

Saccharomyces cerevisiaeand Neurosporacrassa have been studied for absorping of mineral ions.

Extensively studied of by using radioisotopes.

Due to methods problem, the study of ammonium ions have been hinder even though it plays important roles in metabolism and metabolic regulation.

Divalent cations in transport mechanisms have been examined to far lesser extent.

GROUP 6NUR AMIRAH BINTI SIDEK (164673)

SYUKRIYAH BINTI MAT DAUD (162549)SITI NUR ALIA BINTI RAMLI (162568)

CHOO KIN YAN (163668)LEE SIEW YI (163649)

SHEIK ABDUL MUIZZ BIN SHEIK IBRAHIM (162386)MUHAMMAD ADIB AMIN BIN AZHAN (164865)

PotassiumAmmoniumDivalent cations and ironAnion transport

POTASSIUM

POTASSIUM

1. Mechanism transport for Potassium.• Active transport by using permease.• Direct participation of H+-ATPase.• Passive transport by involving Ion

channel.

2. Active transport by using permease.• Active absorption of potassium

occurred depending on the presence of fermentable or respirable substrate.

• The absorption kinetics demonstrated substrate saturation at high concentration following the Mechealis-Menten equation indicating the occurrence of a potassium permease.

3. Direct participation of H+-ATPase. Other alkali metal ions were transported but with

less affinity for the carrier in the order K+>Rb+>Cs+>Na+>Li+ with relative ratios 100:42:7:4:0.5.

H+ and K+ were about equal in the inward transporting system but the outward transporting system discriminated against K+ in favor of H+ or Na+

4. Passive transport by involving the iron channel.

The presence of K+ channels and ion channels sensitive to mechanical stimuli provide additional mechanisms for K+ transport.

Ion channels are a class of plasmalemma proteins that function as gated pores that allow ions to flow down electrical or chemical gradients.

• Ion channels that appear in the membrane patches in response to mechanical deformation of the membrane are called mechanosensitive or stretch-activated channels.

• Strectch activated channels have also been proposed to function in tugor regulation.

• Ion channels did not function in active transport, but their presnce adds another factor to consider in the overall transport equation.

AMMONIUM

AMMONIUM

Ammonium ion has been studied by using methylammonium labeled wit 14C otherwise there was no convenient method for studying this ion.

The use of the methylammonium was difficult due to its toxicity to most fungi.

Both of the ions are using the same transport system because the observation shows that ammonium ion was a potent competitive inhibitor of methylammonium transport.

In S. cerevisiae ,methylammonium transport occur by a typical carrier-mediated, active transport.

In P. chrysogenum and A. nidulans indicated the presence of a repressible high –affinity system for methylammonium transport that was competitively inhibited by ammonium but unaffected by amino acid which is contrast in the S. cerevisiae.

DIVALENT CATIONS AND

IRON

DIVALENT CATIONS

Divalent cations( Mg²+, Ca²+, Mn²+) will be taken up by carrier-mediated transport system.

Transport occur:1. Simultaneously, with phosphate up take.2. Starved cell will firstly interact with

glucose and phosphate. Potassium will greatly simulate the

transport. Dinitrophenol, azide, and arsenate inhibit it.

FE³+ TRANSPORT

Involve siderochromes; Excreted under iron-deficient condition. Involved in active transport. Aspergillus, Neurospora, Ustilago and

bacteria. Against concentration gradient. Competitive inhibition occur between related

siderochromes as a means to stop the intake of iron when it became saturated.

DIFFERENT FUNCTIONS OF FERRICHROME AND FERRICHROME A

FERRICHROME FERRICHROME A

Found intracellular in Ustilago maydis.

Both iron-sufficient and iron deficiency conditions.

Secreted outside. Iron deficiency

only. Solubilizing agent,

to solubilize iron.

ANION TRANSPORTPhosphate, sulfate, nitrate, and organic acids.

ANION TRANSPORT1. Phosphate

Carrier-mediated. Depend on exogenous fermentable

substrate. Phosphate transport acts as

hydroxide exchange system based on observation of the pH change in the medium.

Inhibit by arsenate.2. Sulphate

Study in filamentous fungi. 2 system

ANION TRANSPORT

3. Nitrate Lack study due to the of suitably sensitive

measuring techniques. Diffuse through the membrane in response

to concentration gradient created by nitrate reductase.

GROUP 7

SUGAR AND AMINO ACID TRANSPORT

ADENOSINE TRIPHOSPHATE (ATP)

The main energy source for active transport of sugar.

The energy cost is one ATP per sugar molecule.

Fermentation of sugar, e.g: glucose produces ATP.

DIFFERENCES BETWEEN ACTIVE AND FACILITATED TRANSPORT

Active transport Facilitated difussion

Required ATP for transportation of molecule.

Does not required ATP, but used the concentration gradient (external > internal) to transport molecule.

**Similarity :Both mechanism use carrier proteins to transfer molecules to and from the cell.

SUGAR TRANSPORT

The fungi that utilised facilitated diffusion:1. Saccharomyces cerevisae2. Neurospora crassa

The fungi that used active transport:1. Rhodotorula gracilis2. Aspergillus nidulans

• The glucose transport in Saccharomyces cerevisae is controlled by two systems: Low affinity constitutive system High affinity repressible system

• The inhibitor of glucose transport in S. cerevisae: Uranyl ion, (UO2)2+.

**Others inhibitors which affect transport of some sugar: 2,4-dinitrophenol Azide Carbonylcyanide-m-chlorophenylhydrazone

(CCCP)

IN SACCHAROMYCES CEREVISAE Glucose and galactose transport systems required

kinase activities and transport proteins. Kinase activities: phosphorylation

A glucose transporter was associated with SNF 3 locus while a galactose transporter with the GAL 2 locus.

The SNF 3 and GAL 2 proteins were found in the plasmalemma.

Glucose transport Galactose transport

The enzymes involve in the kinase activities:1. Hexokinase 12. Hexokinase 23. Glucokinase

The enzyme:1. Galactokinase

AMINO ACID TRANSPORT

• Stage of development• Age of cultures• Medium contents

MULTIPLE TRANSPORT SYSTEM WITH OVERLAPPING SPECIFICITY

Table below summarizes the amino acid transport systems of Neurospora crassa

System Amino Acid Specificity1 Aromatic and aliphatic2 Aromatic, aliphatic and

basic3 Basic4 Acidic5 Methionine

SPECIFIC TRANSPORT SYSTEM FOR SINGLE AMINO ACID

This system has been identified in P. chrysogenum and Achlya.

In S. cerevisiae, it has single general amino acid permease (GAP) with broad affinity, basic amino acid transporter, and a series of specific transporters for individual amino acids.

Fungi seem to be quite individualistic in the organization of their amino acid transport abilities.

Amino acid transport of fungi is an active process.

Anoxia and cyanide are the transport inhibitors in most fungi, showing that endogenous respiration is necessary. However, S. cerevisiae is capable of anaerobic transport.

In P. chrysogenum, uncoupling agents inhibit amino acid transport, but arsenate (phosphate competitor) does not inhibit the transport.

Transport Inhibitors

MOLECULES INVOLVED IN THE TRANSPORT PROCESS

Several kinds of evidence suggest that amino acid binding molecules involved in this process.

These include the demonstration that:1. The molecules are associated with the cell

surface or part of the membrane.2. They have the same specificities as the

transport systems. 3. Their removal results in decreased transport

ability of the fungus.4. They are reduced, absent, modified in

transport-deficient mutants.

THE END