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Metabolic Regulation > Shepherd > Flashcards

Shepherd Flashcards

0
Q

Ammonia utilisation: Anammox reactions

A

NH4+ + NO2- –> N2 + 2H2O + energy
N2H4 in anammoxosome which has ladderanes which help to stop diffusion.
4H+ produced for ATP synthesis

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1
Q

The nitrogen cycle

A

Pool of biologically available nitrogen
NH4 in clay -> nitrate NO3- used by plants
NO3- -> N2 in water logged fields
NO + O3 -> nitric acid/ acid rain

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2
Q

Nitrogen fixation (root nodules)

A

Rhizobium in root molecules of plants has a mutualistic relationship.
Nitrogen -> ammonia is favourable
Triple N-N bond requires high energy to break

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3
Q

Haber Bosch process
Biological conditions
Equation

A

Synthetic ammonia was used for the production of HNO2
150-250 bar and 300-550oC
Biological fixation at 0.8bar and biological temp
High activation energy reduced by ATP
N2 + 10H+ + 8e + 16ATP –> 2NH4 + 16ADP + 16Pi +H2

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4
Q

Biological enzymes for ammonia production

Nitrogenase complex structure

A

Nitrogenase complex- dinitrogenase reductase and dinitrogenease.
DR- dimer of 2 identical subunits, 4Fe-S cluster and ATP binding site on each subunit.
R- 2 types of cofactor. 2 P clusters made up of 4Fe-4S clusters. 2 Fe-molybdenum cofactors (unknown X) also containing S and homocitrate

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5
Q

Nitrogen fixation by nitrogenase complex

Electrons

A

Ditrogenase reductase- reduces dinitrogenase
Additional 2e used to 2H+ -> H2
8e required per N2 molecule
Binding of 2ATP -300 -> -420

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6
Q

E flow in nitrogenase reaction

A

Only nitrogenase reductase with 4-Fe4-S in +1 and 2MgATP can associate with nitrogenase (MoFe protein).
2MgATP + e from 4Fe4S –> 2MgADP + e accepted by MoFe.
8 turns of cycle = 2 ammonia
8e = 2 for H2, 6 for 2x NH3

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7
Q

The FeMO cofactor

A

Site of substrate binding and reduction
Strains deficient are inactive, but reactivated by adding FeMo
In CO inhibited FeMo, shows that CO binds to FeMo cofactor, no to the P cluster

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8
Q

The P cluster (nitrogenase)

A

Mediate electron transfer between Fe proteins and the substrate reduction site of the FeMo cofactor
p cluster is between 4Fe-4S and the cofactor
AA substitutions between P cluster and FeMo disturb electron transfer.

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9
Q

Nitrogenase complex is oxygen labile

A

Ditrogenase has a half life of 10 mins
Some bacteria use a respiratory oxidase to burn O2.
Symbiotic supplies nitrogen and bacterial requirements
Engineering of transgenic plants and bacteria to fix N2 (no fertiliser).

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10
Q

Leghaemoglobin

A

Haem binding protein that scavenges oxygen from bacteroid.

Similar to myoglobin, haeme cofactor.

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11
Q

Functions of NO

A

Relaxes smooth muscle and acts as a vasodilator.
Activates guanylyl cyclase which makes cGMP
cGMP PK -> myosin light chain phosphatase -> muscle relaxes

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12
Q

NO is toxic to bacteria

A

Reacts with haeme groups, destroys iron clusters
Can form SNOS and nitrosyl heme
Respiratory inhibition and death
NO + superoxide -> peroxynitrate which can nitrate Tyr residues.

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13
Q

Bacteria encounter NO during infection

A

Nitrate + stomach acid -> NO
Anaerobic respiration -> NO
iNOS and eNOS
Arg -> hydroxyarginine -> citrulline + NO

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14
Q

What triggers iNOS to release NO?
What are the 3 subunits?
What is the reaction?

A

Gram -ve covered in LPS, triggers iNOS
1- haeme prosthetic group and BH4
2- FMN and FAD-NADPH as cofactor
3- calmodulin binding group so Ca sensitive
Arginine -> hydroxyarginine->citrulline + NO

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15
Q

Mechanism for NO synthases

A

cGMP blocks Ca entry to the cell
Ca increases Arg + O2 -> NO
NO then increases cGMP levels
iNOS binds calcium permanently

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16
Q

Bacterial adaptations to NO toxicity

A

NO -> nitrate by Hmop
NO -> N2O via NorVW
NO is reduced by NrfA
YtfE repairs damaged FeS clusters damaged by NO stress
CydAB allows aerobic respiration under low O2

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17
Q

Hmp detoxifies NO

Equations

A

HMP-Fe (2)O2+ NO -> HMP-Fe(3) +NO3-
2HMP-Fe (2)NO + 2H+ –> 2 HMP-Fe(3) + N2O + H2O

Catalysed by Fe haeme cofactor
E shuttled from NADH via FAD to reduce iron

18
Q

Hmp mechanism debate

A

Debate over O2 first or NO first

May depend on availability of O2 and NO

19
Q

Flavohaemoglobin regulation

A

NsrR transcription regulator represses Hmp in low NO
With NO, FeS cluster is nitrosylated and dissociates from DNA
Hmp is transcriptionally regulated by Hcy.
NO -> Inactivates Hcy -> more Hmp expression by metR
NO + 4FeS –> iron species relieve repression.

20
Q

What is bNOS?

A

Bacterial NO synthase, gram +ve
Lacks redoxase domain
Probiotic bacteria offer continuous NO supply useful for research

21
Q

How bNOS increases antibiotic resistance

A
  • chemical modification of toxic compounds by NO
  • alleviation of oxidative stress by antibiotics
    NO suppresses ROS produced by antibiotics
    Inhibition of bNOS research?
22
Q

Bacteria use hydroxamate to import iron

A

Siderophores bind iron and transport
Hydroxamate groups strongly bind Fe3+
Iron is then reduced so is useful to cell
Hydroxamate is recycled

23
Q

What are enterobactins?

A

Derivatives of catechol
Bind ion with high affinity via 6 O2 atoms
Complex is imported through FepA channel -> peri plasm
FeB chaperone -> cytoplasm via active transport (ATP)
Fes esterase -> free Fe
TonB gate uses PMF (H+ pumped)

24
Q

FepA structure

A

Monomeric
Beta barrel
N-terminal plug domain

25
Q

Gram -ve bacteria use haeme as an iron source

A

Hemophore transports Heme -> peri plasm
Heme binds chaperone
Chaperone thigh outer membrane via tonB
Uses ATP
Haeme oxygenase converts haeme -> biliverdin + CO + Fe3+
Pore forming α toxin which lyses erythrocytes

26
Q

Regulation of iron uptake genes by Fur

A

Fur- ferric uptake regulator, transcriptional repressor
Low Fe, dissociates allowing transcription
Fe responsive gene transcribed

27
Q

Metal deprivation during S.Aureus infection

A

Neutrophils restrict growth by removing Fe, Mn and Zn.

Known as nutritional immunity.

28
Q

How the host restricts metal

A

Iron binding activity of lactoferrin (Lf) and transferrin (Tf)
Calprotectin (CP) limits zinc and Mn available
Lf and CP are produced by neutrophils

29
Q

S.Aureus competes with the host for Fe

A

Host- in lysis, hemopexin and haptoglobin remove haeme
Bacteria- lack of Fe removes Fur repression of genes
Produces sideophores to scavenge iron
Haemoglobin captured by isd. Catalysed heme extraction.

30
Q

The isd system of Fe extraction

A

Haemoglobin and heme recruited to cell wall via isd receptors
IsdC transports haeme to IsdDEF membrane transport system.
Then degraded by haeme oxygenases

31
Q

S.Aureus induces Zn/Mn responsive genes

A

Transcriptional repressors MntR and Zur
Allow response to depleted environments
Activates MntABC transporters

32
Q

S.Aureus needs metal to combat host defences

A

Without metals, lack of PMF
Unable to deal with NO and ROS
Hmp and catalase
Mn dependent superoxide mutases SodA and SodM

33
Q

Primary metabolites

A

Form during the exponential growth phase
Ethanol is an example, formed in proportion to growth
Glucose -> Pyruvate -> acetaldehyde -> ethanol

34
Q

Secondary metabolites

A

Usually form at the end of exponential growth/ stationary phase

  • not essential
  • dependent on growth conditions
  • group of closely related compounds
  • concentration at max during late stage
  • require many steps to synthesise
35
Q

Control of secondary metabolism

Most well known auto inducers

A

Metabolic precursors -> production of 2ndary metabolites

  • increase amount of limiting precursor
  • inducing a biosynthetic synthase

Most well known are

  • g-butyrolactones
  • n-acylhomoserine (AHLs of gram -ve)
  • oligopeptides of gram +ve
36
Q

Y-butyrolactones of actinomycetes

A
  • specific group of bacteria that resemble fungi
  • inducing effect of a-factor
    Induces formation of
    -aerial hyphae
  • conidia
  • streptomycin synthases
37
Q

A-factor induces antibiotic production

A

Disappears before streptomycin at its max level.
-synthesised from DHAP
Stimulates
AphD, strR, strB

38
Q

A-factor mechanism

A

A-factor + ArpA -> removal of repressor

Allows secondary metabolism and morphological differentiation

39
Q

Homoserine lactones (HSLs)

A

Related to A factor
Excreted by cells then complex binds to promoter controlled by quorum sensing
AHL -> LuxR -> transcription

40
Q

Bacteriocins and lantibiotics

A 
Heat stable peptides effective against other bacteria, specific immunity
Unmodified and modified peptides
-elongated
-phospholipase inhibitors
-inhibitors of wall synthesis
41
Q

Nisin discovery

A

Approved to be used in cheese.

Elongated, amphiphillic and pore forming

42
Q

Lanthionine synthesis and structure

A

Form when Dha or Dhb condenses with the sulphydryl group of a neighbouring Cys
Form loop structures with S-S
Creates nisin

43
Q

Bacteriocins of lactic acid bacteria mechanism

A

Regulated by quorum sensing
Nisin acts as pheromone to induce its own production
ATP binding -> cleaves leader peptide
Pheromone/ bacteriocin recognised by 2-component transduction

Metabolic Regulation (3 decks)

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