M7 - Lipid Nucleotide Arminio Acid and Hydrocarbon Metabolism

Isoprenoids

Lipid molecules with 2+ isoprene units. Found in plastoquinone and ubiquinone (also carotenoids, archaeal lipids plasma membrane etc)

Lipophilic Compounds

Compounds that accumulate into inclusion bodies in the cytoplasm and serve as E and carbon storage. Most are synthesized from polymeric Polyhydroxybutrate (PHB) lipids, while some A/B have triglycerols and wax esters

FA metabolism

FA of various chains and lengths are can be metabolized as some aerobe’s organisms sole source of E and carbon. FA are degraded with Beta-oxidation pathway or used as precursors for phospholipid biosynthesis.

Proteins that help regulate FA uptake and regulation

FadL

FadD

FadR

FadL

Protein that functions as a ligand gated channel to allow the transport of lipids into a cell

FadD

Protein that functions as Fatty Acid acyl-CoA synthetase that activates FA by converting them into FA-CoA.

FA-CoA

Activated form of FA that binds to FadR, inactivating it.

• Allows activation of biosynthetic pathways

FadR

Regulatory protein that uses FA-CoA as a ligand.

• With FA-CoA, unable to repress transcription

• W/out FA-CoA, represses transcription

Processes FadR regulates (5)

1 FA transport (fadLD)

2 FA catabolism (fadEFGBAH)

3 Universal Stress Protein (uspA)

4 Glyoxylate Shunt (iclR)

5 Unsaturated FA biosynthesis (FabA&B)

FadD (FA-CoA synthetase)

ATP dependent enzyme that catalyzes the addition of a CoA group to FA.

• “requires 2” ATP

• resulting AMP is later converted to ADP with ATP

Beta-Oxidation of Fatty Acids (4)

Pathway that Generates large number of reduced cofactors (NAD, FADH2, and Acetyl-CoA) that go to TCA

Common in aerobes

Basically a Cycle

Reactions largely occur on Beta-Carbon

Enzymes in Beta-Oxidation pathway

1 Acetyl-CoA dehydrogenase

2 Enoyl-CoA Hydratase

3 Beta-Hydroxyl acyl CoA Dehydrogenase

4 Beta-keto thiolase

Reactants/Products in Beta-oxidation (5)

1 Fatty-acyl-CoA

2 trans delta2 fatty enoyl-CoA

3 L-Beta-Hydroxy Acyl-CoA

4 Beta-keto acyl-CoA

5 FA-CoA and Acetyl-CoA

Propionyl Acetyl-CoA (3)

3C structure generated if acyl chain is odd

Used in amino acid biosynthesis (Ile, Val)

Can enter metabolic pathway (acrylyl-CoA, methylcitrate or methylmalonyl-CoA)

Beta-Oxidation of Palmitic Acid

oxidation pathway that yields 8 ATP from SLP and 123 ATP from OxPhos

Types of Hydrocarbon metabolism (2)

Aerobic Pathways

Anaerobic Pathways

Aerobic Hydrocarbon Pathways

Hydrocarbon metabolism pathway common to fungi and B

Require O2 and membrane-bound monooxygenases

Can be used to clean up oil spills

Anaerobic Hydrocarbon Pathways

Hydrocarbon metabolism pathway linked with SO4-2 reduction

Can cause oil well, souring, and corrosion

Monooxygenases

O2 dependent enzymes that cat oxidation of co-substrate and hydrocarbons.

Use Co-substrate rubredoxin, cytP450, or combnty***

Hydrocarbon n-Alkane Metabolism pathways (2)

1 Terminal Oxidation pathway

2 Subterminal Oxidation Pathway

Terminal Oxidation pathway

Oxygen-dependent monooxygenase used with rubredoxin to metabolize alkanes.

Terminal alkane has hydroxyl group on terminal end

Products move to Beta-oxidation pathway

Steps in Terminal Oxidation pathway

1 rubredoxin: NADH oxidoreductase

2 n-alkane monooxygenase

3 Primary Alcohol dehydrogenase

4 Aldehyde dehydrogenase

Subterminal Oxidation Pathway

Uses 2 monooxygenases that aren’t O2 dependent

Initial monooxygenases introduce hydroxyl group subterminal

Generates acetate and more reduced cofactors

Products continue to beta-oxidation

Steps in Subterminal Oxidation Pathway

1 Monooxygenase 1

2 Secondary alcohol DH

3 Monooxygenase 2

4 Esterase

5 Primary alcohol DH

Terminal Oxidation Pathway Overall Reaction

HC + 1AH2 → 2NADPH + FA

Subterminal Oxidation Pathway Overall Reaction

HC + 2AH2 → 3NADH + Acetate + FA (-2C)

Acyl Carrier Protein (ACP) (6)

Involved in initial FA biosynthesis

Small Protein

carries acyl group that’s attached to FA

Activated when phosphorylated

Serine residue is phosphorylation target

Co-A-Like

PPTase (3)

Involved in initial FA biosynthesis

Phosphorylates ACP to activate it

CoA is substrate

FA Biosynthesis Pathway (3)

Cyclical pathway that converts acetyl-CoA into acyl-SACP

acyl-SACP (product) may be turned into acetyl-ACP (Earlier intermediate) (Malonyl-Carbon-CoA)

Requires ATP, 2NADPH, and HCO3

Produces C16-18 (or smaller) molecules

Fatty Acid Biosynthesis Pathway Steps (enzymes)

1 ATP-Dep Acetyl-CoA Decarboxylase

2/3 Transacetylase

4 Synthetase

5 Reductase

6 Dehydrase FabA

7 Reductase

FA Biosynthesis Pathway Intermediates and Products

0 Acetyl-CoA

1 Malonyl-CoA

2 Malonyl-ACP

3 Acetyl-ACP (Malonyl-carbon-CoA)

4 3-Ketoneyl-SACP

5 3-OH acyl-SACP

6 enoyl~SACP

7 acyl~SACP

Malonyl-CoA formation (3)

First committed step in FA biosynth

Frist step of FA biosynth

Catylized by ATP-Dep Acetyl-CoA Decarboxylase

Unsaturated FA biosynth

Unsaturated FA are created by introducing db into chains

O2 dependent

O2 independent

Regulation of Unsaturated FA biosynth

Signals that alter membrane fluidity tell cell to increase of decrease fluidity

• low temp → signals to increase fluidity

• Bioproducts: ethanol → signals to decrease fluidity (makes more stable for bioproducts)

O2 Dependent Unsaturated FA Biosynthesis (3)

Desaturases used to introduce cis/trans bonds into FA

Uses O2 to draw e- from chain

Modification of a complete acyl-ACP molecule

Desaturase

Catalyzes formation of cis/trans db in FA

Requires O2 and NADPH

Integral membrane

Transfers 2e- from acyl group and 2e- form NADPH to reduce O2 to H2O

O2 Independent Unsaturated FA Biosynthesis (4)

Introduction of a db to a 10C or 12C chain during FA production/elongation

If db trans, saturated

If db cis, unsaturated

Fab Proteins used to convert between cis/trans conformation

Phospholipid biosynthesis

DHAP (Dihydroxyacetone phosphate) is eventually converted to CDP-diacylglycerol, which is used as a starting point to produce many phospholipids.

Involves products from central metabolism (DHAP) and FA biosynth (acyl-ACP/-CoA )

Steps/enzymes in Phospholipid Biosynth (4,3)

1 Glycerol-3-phosphate dehydrogenase (GPDH)

2 Acetyl Transferase

x Phosphatidic Acid

3 CDP-Diacyl-glycerol synthase (CDS)

Phospholipid Biosynthesis Step 1 (3)

DHAP is converted to G3P by GPDH (Glycerol-3-phosphate dehydrogenase).

Requires input of e- from NADH

DHAP is input from central metabolism

Phospholipid Biosynthesis Step 2 (3)

Acetyltransferase catalyzes the conversion of G3P to PA with the de-acetylaiton of acyl-ACP/CoA.

HIGHLY regulated

Inhibited by STARVATION

Phospholipid Biosynthesis Step 3 (4)

DP-diacylglycerol synthase (CDS) converts Phosphatidic Acid (PA) into CDP-diacylglycerol through the attachment of a CMP group.

Generates CDP-diacylglycerol, a high E intermediate (“ready for Nu attack")

Activates PA

CTP dependent

Phosphatidic Acid (PA)

Intermediate in and “Parent compound” for phospholipid metabolism

CDP-diacylglycerol ()

Key precursor for many phospholipids

Conversion/processing is cat by membrane bound enzymes

Has positivly charged and endophlilic phosphates (ready for Nu Attack)

S-Adenosyl Methionine (SAM) (4)

Coenzyme

Synthesized from Met and ATP

C1 Carrier for donation of methyl groups

Methylates when nucleophile attacks methyl group

Methionine acetyltransferase

Converts Met to SAM coenzyme with ATP

Regeneration of SAM

SAM is regenerated by cooperating methionine and folate cycles

Steps in SAM regeneration

1 5-Adenyl Homocysteine (5AH) is hydrated to adenosine and homocysteine (HCY)

2 HCY is subject to VB12-dep RXN, regenerating MET

3 Tetrahydrofolate (THF) accepts a C from serine, converting to 5,10-MTHF

4 5,10-MTHF is reduced to 5-MTHF, which can then donate the methyl to HCY in VB12 RXN

Tetrahydrofolate (THF)

C1 carrier in the folate cycle

Has pteridine ring that accepts the CH3 group (5-foryl THF)

Has p-ABA group that accepts an alt CH3 group (10-foryl THF)

Sulfa Drugs

Drugs that prevent the Folate cycle by mimicking pABA (p-aminobenzoic acid) that makes folate

Inhibit folate biosynthesis, THF cycle, and purine biosynthesis

Bacteriostatic agents

Flippase (3)

Move phospholipids from outer to the inner membrane

ATP-dep

P4ATPase Family

(Flip → moves in )

Floppase (3)

Moves phospholipids from inner to outer membrane

ATP-dep

ABC Family

(Flop → moves out)

Scramblase

Moves phospholipids back and forth across membranes down their [gradient]

Ca+2 dep => E independent

(Evolutionarily Distinct Protein Families)

Lipid Transport Between Membranes - Gram Negatives

Gram negatives must flip lipids and transport them between inner and outer membranes

AsmA like proteins

MIa proteins

AsmA like proteins (4)

Proteins that move lipids from inner to outer membrane in gram negatives

Have hydrophobic groove or “bridge” that shields lipids in the periplasmic space

Mediate lipid transport between organelles

Related to eukaryotic Beta-groove protein family

Archaeal Lipids

Have branched isoprene chains

Isoprenoid Biosynthesis

Common pathway in A that produces Isoprenoid Lipids (quinones, carotenoids, sterols, etc)

Two pathways

• Mevalonate pathway

• DXP pathway

Mevalonate pathway (4)

Pathway to produce isoprenoid lipids in A & E

Starts with Acetyl-CoA and includes mevalonate as key intermediate

HMG-CoA reductase is rate limiting step/Ez

Target of cholesterol lowering drugs

Dxp Pathway

Pathway to poroduce isoprenoid lipids in bacteria

No HMG-CoA reductase

Has Deoxy-xylose-phosphate (DXP) intermediate

Diversity in mevalonate pathways

Pathways branch heavily after production of mevalonate

Intersects with A biosynth of phospholipids (g-1-p backbone)

No ACYL-CARRIER protein

Pyrimidine Nucleotide synth (4)

THF-INDEPENDENT

E-dep Synthetase domain incorporates Carbon from bicarbonate

Glutamase domain incorporates nitrogen from ammonia

Asparate provides remaining 4C backbone

Carbamoyl Phosphate Synthetase (CPS) (4)

Ez that initiates Pyrimidine synthesis

Type varies based on N donor (NH3 vs Gln)

Needs allosteric effector N-acetyl-l-glutamate for activity

Have glutaminase and synthetase domain

Inhibitors of Carbamoyl Phosphate Synthetase (CPS)

ornithine, IMP, and UMP

N-acetyl-l-glutamate

Activates Carbamoyl Phosphate Synthetase for Pyrimidine biosynth

Aspartate transcarboxylase (ATCase)

Ez that cat the 2nd step in Pyrimidine biosynthesis

Combines Asparate and CP into carbamoyl asparate (makes backbone)

Feedback Inhibited

Aspartate transcarboxylase (ATCase) INHIBITORS

Inhibited by CTP

Feedback inhibition

(inhibitors designed have applications in treating bacterial pathogens, malaria, oncology, immunosuppression, etc.)

Pyrimidine Nucleotide Biosynthetic Pathway Steps (Ez)

1 Carbamoyl phosphate synthetase

2 Aspartate transcarbamoylase

3 Dihydroorotase

4 Dihydroorotate dehydrogenase

5 Orotate phosphoribosyltransferase

6 Orotidylate decarboxylase

7 CTP synthetase

PRPP Synthetase (PRS) (4)

key biosynthetic precursor ENZYME for Pyrimidine, Purine, & AA pathways

Ribose-P-pyrophosphokinase

ATP-dep conversion of Ribose-5-Phosphate → PRPP (phosphoribosyl pyrophosphate)

Feedback inhibition

PRPP Synthetase (PRS) inhibitors

Feed back inhibited by AMP, ADP, GDP (LOW cellular E)

PRPP product

key precursor for purine, pyrimidine, NAD+/NADP+, and amino acid (His, Trp) biosynthesis

Purine biosynthesis (4)

More complex biosynthesis due to double ring structure.

Derived from G, D, Q, CO2, and C1 units (from formate or serine)

THD-dependent

INHIBITED by SULFA durgs

Purine Nucleotide Biosynthetic Pathway (4)

Early attachment of PRPP

Initial steps feedback inhibited by purine nucleotides

• Step 1 = PRPP synthetase

• Step 2, PRPP aminotransferase

Tetrahydrofolate (THF)-dependent

Inosine monophosphate (IMP, containing hypoxanthine) is a key intermediate

• Produced at the end

Purine Nucleotide Biosynthetic Pathway Feedback INHIBITION

Feedback inhibition by ATP and GTP

Early steps also tightly regulated

Inosine monophosphate (IMP)

Key intermediate produced from Purine biosynthesis

Can be used as precursor for ATP and GTP biosynthesis

Feedback inhibition by ATP and GTP

Ribonucleotide Reductase (RNR)

Cat the conversion of RNA building blocks to DNA building blocks

• ribose sugar of nucleotide diphosphate (NDP) to deoxynucleotide diphosphate (dNDP)

• eduction of 2'OH-group

Thymidylate Synthase Pathway (4)

Pathway that methylates dUMP to dTMP

Involved in DNA synth

Antibiotic and chemotherapy target

Involves THF

Nucleotide Salvage

Base specific phosphoribosyltransferase salvage/recycle bases from nucleic acid (RNA/DNA)

turnover for use in biosynthesis

DNA/RNA Catabolism

When nucleic acids are used as a carbon and Nitrogen source (all domains)

Common to break down urea to extract purine/pyrimidine (common waste product)

• Nucleotidase

• nucleosidase

• nucleases

nucleases

Ez that breaks down DNA/RNA polymers

Nucleotidase

Ez that dephosphorylates nucelotides, releasing Pi/phosphate from purine/pyrimidine bases

nucleosidase

Ez that release ribose and deoxyribose from nucleosides (purine/pyrimidine bases)

Glutamate and Glutamine in AA biosynth

AA that provide amino groups for biomolecules and AA

→ amino group donor and acceptor pair

transamination rxns

Catalyze the donation of an amine group form an amine group donor (like Glu) to form AA (or other biomolecules)

Gln

serve as a N-donor for His, Trp, Asn, asparagine, purines, pyrimidines, amino sugars, and NAD+

Ammonia Assimilation at High [NH3]

glutamate dehydrogenase and aminotransferase catalyze the assimilation of NH3 into AA (or other biomolecules)

glutamate dehydrogenase

Enzyme with LOW affinity for NH3

Catalyzes the reductive assimilation of NH3 into Glu

Oxidizes NADPH to NADP+ (requires NADPH for energy)

Reversable

aminotransferase

Cat the deamination of AA of amine donor

OR

Cat the amination of a keto-acid, ketone, or aldehyde (AA aceptor)

Reversable

GS/GOGAT Pathway

Pathway that catalyzes Ammonia Assimilation at Limiting [NH3]

Ammonia Assimilation at Limiting [NH3]

Glutamine synthetase, Glutamine: 2-oxoglutarate Aminotransferase, and aminotransferase are used to catalyze the assimilation of NH3 into AA

Glutamine Synthetase (GS)

HIGH affinity for NH3

Catalyzes the ATP-dep assimilation of NH3 into Gln, glutamine

ATP-dependent reaction

Essential for growth (unless cells supplemented with Gln)

Glutamine: 2-oxoglutarate Aminotransferase (GOGAT)

Cat NADPH dep conversion of Gln to Glu

Synthesizes 2 mol glutamate from glutamine and α-ketoglutarate via aminotransferase reaction

Pyridoxal 5′-phosphate (PLP)

Key cofactor for AA biosynthesis

Active form of VB6 involved in transamination

Binds to transaminase and acts as amine carrier

Amino Acid Catabolism

Catabolism of AA initiated by deamination RXNS to remove amino group

Generates alpha-Keto acids

Different breakdown for glucogenic and ketogenic AA

Glucogenic AA (catabolism)

AA can be converted into glucose through gluconeogenesis

Ketogenic AA (catabolism)

AA that can be broken down metabolically and converted into acetyl-CoA

Ile, Leu, Thr, Trp, Tyr, Phe, Lys

(“I’lL TrY Wed Fry weeKdays”)

β-hydroxybutyrate (BHB)

A ketone body that serves as an energy source (for ketogenic AA) as resources become limiting

Acetyl-CoA is a precursor

L-amino Acid Catabolism

AA catabolism

Initiated by aminotransferases (primarily, shown earlier slides) or deaminases, depending on the amino acid

D-amino Acid Catabolism

AA catabolism

Initiated by D-amino acid oxidases (DAAO), which catalyze oxidative deamination