Download 12.1 Mechanisms regulating enzyme synthesis 12.1.2.2 Enzyme

발효화학 (Fermentation Chemistry), Bacterial Physiology and Metabolism
Chapter 12. Metabolic regulation
Yong-Cheol Park
(http://park.openwetware.org)
Department of Advanced Fermentation
Fusion Science & Technology
Introduction
 Microbial ecosystems are oligotrophic with a limited availability of nutrients.
 Furthermore, nutrients are not usually found in balanced concentrations while
the organisms have to compete with each other for available nutrients.
 Organic materials are converted to carbon skeletons for monomer and polymer
synthesis, as well as being used to supply energy.
 This is possible through the regulation of the reactions of anabolism and
catabolism.
 Since almost all biological reactions
are catalyzed by enzymes,
metabolism is regulated by
controlling the synthesis of enzymes
and their activity (Table 12.1).
 In this chapter, different mechanisms
of metabolic regulation are discussed
in terms of enzyme synthesis through
transcription and translation and
enzyme activity modulation.
12.1 Mechanisms regulating enzyme synthesis
12.1.2 Induction of enzymes
12.1.2.1 Inducible and constitutive enzymes
 Enzymes synthesized in the presence of substrates are referred to as inducible
enzymes and the substrate is termed the inducer.
 Inducible enzymes are generally those used in the catabolism of carbohydrates
such as polysaccharides (cellulose, starch, etc.), oligosaccharides (lactose,
trehalose, raffinose, etc.) and minor sugars (galactose, xylose, arabinose and
rhamnose, etc.).
 Constitutive enzymes are those enzymes that are produced under all growth
conditions.
12.1 Mechanisms regulating enzyme synthesis
12.1.2.1 Inducible and constitutive enzymes (continued)
 When a single inducer induces more than two enzymes,
(1) they are produced either simultaneously  coordinate induction
(2) or sequentially  sequential induction
 Genes of coordinate induction are in the same operon, and genes from separate
operons are induced sequentially.
12.1 Mechanisms regulating enzyme synthesis
12.1.2.2 Enzyme induction
 Enzyme induction is regulated at the transcriptional level.
 Lactose induces the production of β-galactosidase, permease and transacetylase.
 Their structural genes form an operon (lac operon) with a promoter and
operator.
12.1 Mechanisms regulating enzyme synthesis
12.1.2.2 Enzyme induction (continued)
 The regulatory gene (lacI) next to the 5’ end of the operon is expressed
constitutively with its own promoter.
 In the absence of the inducer, the LacI protein binds the operator region of the
lac operon, inhibiting RNA polymerase from binding the promoter region.
 When the inducer (lactose or IPTG, an analogue of lactose) is available, it binds
the LacI protein, removing it from the operator region.
 In a lacI mutant, the lactose-metabolizing enzymes are expressed constitutively
in the absence of the inducer (lactose or IPTG).
 In this sense, the regulation by repressor proteins is referred to as negative
control.
12.1 Mechanisms regulating enzyme synthesis
12.1.2.3 Positive and negative control
 Negative control: target gene expression without a regulator protein (repressor)
 Positive control : target gene expression with a regulator protein (activator)
 Activator proteins are involved in the regulation of catabolic genes for arabionse,
rhamnose, maltose and others.
 Among the genes for
arabinose catabolism,
araC is a regulatory gene
encoding an activator
protein.
 AraC mutants are unable
to use arabinose, since
an AraC complex with the
inducer activates the
transcription of the
structural genes.
12.1 Mechanisms regulating enzyme synthesis
12.1.2.3 Positive and negative control (continued)
 The term ‘regulon’ is used to define genes of the same metabolism controlled
by the same effectors scattered around the chromosome, such as ara genes.
 Regulation by an activator, as in the ara regulon, is referred to as positive
control.
12.1 Mechanisms regulating enzyme synthesis
12.1.3 Catabolite repression
 When Escherichia coli or Bacillus subtilis is cultivated
in a medium containing glucose and lactose, they
grow in a distinct two-phase pattern (Fig.12.5).
 This is called diauxic growth or diauxie.
 The reason for the diauxic growth is that the readily
utilizable glucose and its metabolites repress the
utilization of lactose.
 In G(-) bacteria, there are two-separate catabolite
repression mechanisms,
(1) one involving a cAMP-CRP (cAMP receptor protein)
complex and
(2) the other a catabolite repressor/activator (Cra)
protein
 In G(+) bacteria, the catabolite control protein A
(CcpA) has a similar function.
12.1 Mechanisms regulating enzyme synthesis
12.1.3 Catabolite repression
12.1.3.1 Carbon catabolite repression by the cAMP-CRP complex
 The primary carbon catabolite repression (CCR) in G(-) bacteria is related to the
intracellular concentration of cyclic AMP (cAMP).
 When the readily utilizable substrate (e.g. glucose) is exhausted,
 the cAMP concentration increases intracellularly
 cAMP makes a complex with the cAMP receptor protein (CRP or catabolite
activator protein, CAP)
 This complex controls many operons (Sec.12.2).
<cAMP synthesis>
 Glucose is transported through PTS in many bacteria.
 When glucose concentration is low,
 enzyme IIA of PTS remains phosphorylated.
 the phosphorylated EIIA activates adenylate cyclase
, a cytoplasmic membrane enzyme.
 This enzyme converts ATP to cAMP.
12.1 Mechanisms regulating enzyme synthesis
12.1.3.1 Carbon catabolite repression by the cAMP-CRP complex (continued)
<cAMP hydrolysis>
 A cytoplasmic enzyme, phosphodiesterase, hydrolyzes cAMP, but the activity is
very low:
 When the level of phosphoylated enzyme IIA is low,
 adenylate cyclase activity is low  the cAMP concentration is kept low
 CRP cannot form the complex.
<cAMP-mediated gene activation>
 When the rate of cAMP formation is higher than that of its hydrolysis, cAMP-CRP
complex formation is facilitated.
 This complex activated the transcription of many operons including the lac operon.
 The cAMP-CRP complex regulates the expression of over 200 proteins.
 Carbon catabolite repression (CCR) by the cAMP-CRP complex is known only in
G(-) bacteria.
 This regulatory mechanism is an example of global regulation (Sec.12.2).
12.1 Mechanisms regulating enzyme synthesis
12.1.3.1 Carbon catabolite repression by the cAMP-CRP complex (continued)
<lac operon>
 When lactose is provided with a low level of glucose
 lactose binds the repressor protein (LacI) (Fig.12.3)
 and the cAMP-CRP complex binds to CRP cite of the promoter region
 this action activates the transcription of the structure genes (Fig.12.6).
12.1 Mechanisms regulating enzyme synthesis
12.1.3 Catabolite repression
12.1.3.2 Catabolite repressor/activator
 In addition of the cAMP-CRP complex, Cra (catabolite repressor/activator) protein functions as
a catabolite repressor and activator in enteric bacteria including Escherichia coli.
 In Cra-negative mutants,
(+) effect : more enzymes of glycolysis and the PTS
(-) effect : fewer enzymes involved in gluconeogenesis, TCA cycle,
glyoxylate cycle and electron transport
unable to utilize non-carbohydrate substrates including acetate,
ethanol, pyruvate, alanine, citrate and malate
<Cra-mediated gene activation>
 With a limited supply of readily utilizable glucose, the Cra protein (1) positively regulates
transcription of enzymes needed for the utilization of other substrates and (2) represses the
genes for glycolytic enzymes (Fig.12.7).
 For example, when acetate or ethanol is used as the substrate, Cra protein activates the
expression of genes for the enzymes of gluconeogenesis (fructose-1,6-bisphosphatase) and
anaplerotic sequence (PEP carboxykinase), and represses the transcription of genes for
glycolytic enzymes (phosphofructokinase).
12.1 Mechanisms regulating enzyme synthesis
12.1.3.2 Catabolite repressor/activator (continued)
 Genes activated by
the Cra protein are
those that have a
Cra-binding site
upstream of the
promoter region.
 Genes are repressed
where the Crabinding site
overlaps the
promoter region or
occupies it
downstream.
12.1 Mechanisms regulating enzyme synthesis
12.1.3.3 Catabolite repression in G(+) bacteria with a low G+C content
 The cAMP-CRP complex is not known in G(+) bacteria which have a different CCR.
 In PTS system of G(+) bacteria, HPr has a second phosphorylation site at serine-46 in
addition to histidine-15.
 PEP phosphorylates the histidine-15 for the PTS while HPr(ser) kinase (PstK) phosphorylates
the serine-46 consuming ATP.
 When glucose is available,
the cell maintains high levels
of glucose metabolites
(fructose-1,6-bisphosphate)
and non-phosphorylated HPr.
12.1 Mechanisms regulating enzyme synthesis
12.1.3.3 Catabolite repression in G(+) bacteria with a low G+C content (cont.)
 Fructose-1,6-bisphosphate activates HPr(ser) kinase to phosphorylate the serine residue of the
non-phosphorylated HPr protein.
 This phosphorylated HPr protein [HPr(Ser-P)] forms a complex with CcpA (catabolite control
protein A).
 The CcpA-HPr(Ser-P) complex binds
to the cre (catabolite responsive
element) located either upstream or
within their promoters.
 This complex represses the
expression of genes for enzymes of
xylose, arabinose, gluconate, glucitol
and mannitol metabolism, and
activates others.
 ATP-dependent HPr(Ser)
phosphorylation is not known in G(-)
bacteria.
12.1 Mechanisms regulating enzyme synthesis
<An example of CcpA-mediated operon>
xylA: xylose isomerase
xylB: xylulokinase
xylR: xyl repressor
: XylR binding site
: ribosome binding site
: CRE
Xylose operon
HPr
PxylA
PxylR
XylR
P
CcpA
Glucose
xylR
- 35
- 10
xylB
xylA
Xylose
XylR
PxylR
PxylA
HPr
P
CcpA
Glucose + Xylose
- 35
xylR
- 10
HPr
Xylose
XylR
PxylR
xylB
xylA
CcpA
PxylA
Xylose
xylR
- 35
- 10
xylA
Transcription of xylAB
xylB
12.1 Mechanisms regulating enzyme synthesis
12.1.3.3 Catabolite repression in G(+) bacteria with a low G+C content (cont.)
 Control by the CcpA-HPr(Ser-P) complex is known in low G + C content G(+) bacteria
including the genera Bacillus, Staphylococcus, Streptococcus, Lactococcus, Enterococcus,
Mycoplasma, Clostridium, and Listeria.
 In Bacillus subtilis, approximately 10% of the genes may be directly regulated by the CcpAHPr(Ser-P) complex
<CRP- vs. CcpA-mediated CCR>
 A difference between the CRP-dependent CCR in G(-) bacteria and the CcpA-dependent CCR
in G(+) bacteria is the strictness of the coupling between the PTS, transport and regulatory
functions.
(1) In the CRP-dependent mechanism, PTS enzyme IIA activates the primary sensor adenylate
cyclase,
(2) In the CcpA-dependent mechanism, HPr(ser) kinase is the primary sensor and activated by
glycolytic intermediates (FBP).
 Metabolic control by the effectors of catabolite repression, cAMP-CRP and CcpA-HPr(Ser-P)
complex, and the Cra protein are examples of global regulation mechanisms (Sec.12.2).
12.2 Global regulation: response to environmental stress
 Though some processes regulate a single operon or regulon, most of them regulate more than one
operon or regulon.
 For example, the cAMP-CRP complex regulates enzymes of more than 200 metabolic processes.
 These regulatory processes are referred to as global regulation, a multigene system or pleiotropic control.
12.3 Regulation through modulation of enzyme activity
 Metabolic regulation through the transcription and translation processes that take time are
inadequate for microbes to cope with a rapidly changing environment.
 For efficient metabolic regulation, enzyme activities are modulated in addition to their
synthesis.
 Enzyme activities are regulated through various mechanisms including feedback inhibition,
feedforward (precursor) activation, and physical and chemical modification of the enzyme
proteins.
 Feedback inhibition:
When an amino acid
is available and
sufficient to meet the
needs of growth, the
enzyme catalyzing
the first reaction is
inhibited.
12.4 Metabolic regulation and growth
 Growth conditions determine the growth rate and
cell yield in a given organism.
 This is due to the differences in energy
conservation efficiency and maintenance energy
when growing on different carbon sources.
 For the most efficient
growth under given
conditions, catabolism and
anabolism are coordinately
regulated not only through
the expression of the gens
but also through the control
of enzyme activities
12.6 Metabolic regulation and the fermentation industry
 ‘Fermentation’ is also used to describe processes producing useful materials to a large
industrial scale using microorganisms.
 Microorganisms have elaborate regulatory mechanism for efficient growth but not for the
production of specific materials.
 To improve fermentation efficiency, industry has developed and uses various mutants
defective in regulatory mechanisms.
12.6.2 Fermentative amino acid production
 Coryneform bacteria of the genera Brevibacterium and Corynebacterium are the most
commonly used industrial strains in amino acid production.
 These bacteria excrete amino acids into the medium when the membrane becomes more
permeable to amino acids under biotin-limited conditions.
 The industrial strains used to produce amino acids are mutant strains with defects in the
various regulation at the gene and enzyme levels.
 These are selected based on their resistance properties to analogues (Table12.9).
 When an amino acid analogue is added to a culture, the wild-type stain cannot grow since
the analogue inhibits expression of the genes for the production of the amino acid and the
analogue cannot be used for biosynthesis.
12.6 Metabolic regulation and the fermentation industry
12.6.2 Fermentative amino acid production (continued)
 Auxotrophic mutants are used for the fermentative production of the intermediates.
 Guanine auxotrophs are used to produce adenine and hypoxanthine.
 Lately, whole genome
sequences have been
determined in some
industrially important
microorganisms and industrial
strains have been developed
through molecular biology
approaches.
 Metabolic engineering aims
to (1) increase enzyme
activities, (2) relieve regulatory
mechanisms and (3) improve
membrane permeability by
modulation of metabolic
genes and enzymes.