Hogg S. Essential microbiology

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148 MICROBIAL METABOLISM

–

870

Red/infrared light

ADP

+ Pi

ATP

NADP

+ H

NADPH

External electron

donors e.g. H

, succinate

ADP + Pi

ATP

Cyt

Bph

Redox

Potential

(V)

Reverse

electron

flow

Figure 6.33 Electron ﬂow in the anoxygenic photosynthesis of a purple bacterium. ATP is

generated by the passage of electrons down an electron transport chain back to the reaction

centre bacteriochlorophyll. Anoxygenic photosynthetic bacteria use molecules such as sul-

phur and hydrogen sulphide instead of water as external electron donors, hence no oxygen

is generated. NADPH for use in CO

ﬁxation must be generated by reverse electron ﬂow.

Bph = bacteriophaeophytin (bacteriochlorophyll a minus its magnesium atom)

Anabolic reactions

So far, in describing respiration and photosynthesis, we have considered those reactions

whereby a microorganism may generate cellular energy from its environment. As we

saw at the beginning of this chapter, one of the uses to which this may be put is the

synthesis of new cellular materials. In all of the pathways described in the following

Table 6.4 Comparison of anoxygenic photosynthetic bacteria

Green Green Purple Purple

sulphur non-sulphur sulphur non-sulphur

Bacteriochlorophyll c, d, ea, ca, ba, b

Electron donor H

S/reduced

S/H

Organic

compounds

S/reduced

S/H

Organic

compounds

ﬁxation Reverse

TCA cycle

Calvin cycle Calvin cycle Calvin cycle

Photoheterotrophy No Yes Some Mainly

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ANABOLIC REACTIONS 149

section, the conversion of ATP to ADP is required at some point. The term biosynthesis

is used to describe those reactions by which nutrients are incorporated ﬁrst into small

molecules such as amino acids and sugars and subsequently into biomacromolecules

such as proteins and polysaccharides.

Biosynthesis of carbohydrates

We have already seen in the preceding pages that autotrophic organisms (not necessarily

phototrophic ones) are able to incorporate inorganic carbon as CO

or HCO

−

into

hexose sugars, most commonly via the Calvin cycle. Heterotrophic organisms are unable

to do this, and must convert a range of organic compounds into glucose by a series of

reactions called gluconeogenesis (Figure 6.34). Many compounds such as lactate or

certain amino acids can be converted to pyruvate directly or via other members of the

TCA cycle, and thence to glucose. To all intents and purposes, gluconeogenesis reverses

the steps of glycolysis (see above), although not all the enzymes involved are exactly

the same. This is because three of the reactions are essentially irreversible, so different

enzymes must be used to overcome this. These reactions are highlighted in Figure 6.34.

Once glucose or fructose has been produced, it can be converted to other hexose

sugars by simple rearrangement reactions. Building up these sugars into bigger carbo-

hydrates (polysaccharides) requires them to be in an energised form: this usually takes

the form of either an ADP- or UDP-sugar, and necessitates an input of energy. Pentose

sugars such as ribose are important in the synthesis of nucleotides for nucleic acids and

coenzymes (see below).

Biosynthesis of lipids

Fatty acids are synthesised by a stepwise process that involves the addition of two-

carbon units to form a chain, most commonly of 16–18 carbons. The starting point of

fatty acid metabolism is the two-carbon compound

The basic building blocks in the synthesis of fatty acids are acetyl-CoA (two-carbon)

and malonyl-CoA (three-carbon). We have encountered acetyl-CoA before, when dis-

cussing the TCA cycle; malonyl-CoA is formed by the carboxylation of acetyl-CoA.

Acetyl–CoA + CO

→ Malonyl–CoA

2 − carbon 3 − carbon

Carbon dioxide is essential for this step, but is not incorporated into the fatty acid as it is

removed in a subsequent decarboxylation step. In order to take part in the biosynthesis

of fatty acids, both molecules have their coenzyme A element replaced by an acyl carrier

protein (ACP). In a condensation reaction, one carbon is lost as CO

and one of the

ACPs is released. The resulting four-carbon molecule is reduced, with the involvement

of two NADPH molecules, to butyryl-ACP. This is then extended two carbon atoms at

a time by a series of further condensations with malonyl-ACP (Figure 6.35).

Thus, extending a fatty acid chain by two carbons involves the expenditure of one

ATP and two NADPH molecules. The overall equation for the synthesis of a 16-carbon

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150 MICROBIAL METABOLISM

Figure 6.34 Gluconeogenesis. Non-carbohydrate precursors can feed into a pathway that

converts pyruvate to glucose in a series of reactions that are mostly the reverse of glycolysis.

Enzymes not found in glycolysis are shown in italics

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ANABOLIC REACTIONS 151

Acetyl-ACP Malonyl-ACP

C C ACP HOOC C C ACP

ACP

C C C C ACP Acetoacetyl-ACP

2NADPH

2NADP

C C C C ACP Butyryl-ACP

Figure 6.35 Fatty acid biosynthesis. Acetyl- and malonyl-ACPs condense with the loss

of CO

to give a four-carbon molecule butyryl-ACP. The addition of further two-carbon

acetyl groups is achieved by re-entering the pathway and reacting with further molecules of

malonyl-ACP

fatty acid such as palmitic acid can be represented:

8 Acetyl-CoA + 7ATP+ 14 NADPH + 6H

−→ Palmitate + 14 NADP

+ 8 CoA +

O + 7 ADP + 7Pi

Once formed, fatty acids may be incorporated into phospholipids, the major form of

lipid found in microbial cells. Recall from Chapter 2 that a phospholipid molecule has

three parts: fatty acid, glycerol and phosphate. These last two are provided in the form

of glycerol phosphate, which derives from the dihydroxyacetone phosphate of glycol-

ysis. Glycerol phosphate replaces the ACP of two fatty acid-ACP conjugates to yield

phosphatidic acid, an important precursor for a variety of membrane lipids. The energy

for this reaction is provided, unusually, not by ATP but by CTP (cytidine triphosphate).

Biosynthesis of nucleic acids

Most microorganisms are able to synthesise the purines and pyrimidines that comprise

the nitrogenous bases of DNA and RNA. These compounds are synthesised from a

number of sources in reactions that require an input of ATP. The contribution of different

compounds towards the purine skeleton of guanine or adenine is shown in Figure 6.36.

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152 MICROBIAL METABOLISM

Figure 6.36 Purine structure. Several precursors contribute towards the formation of the

purine base skeleton. The nitrogen atoms are donated by the amino acids glutamine, as-

partate and glycine. Note the important role played by folic acid. The antimicrobial agent

sulphonamide (Chapter 15) exerts its effect by inhibiting folic acid synthesis, which in turn

affects synthesis of purine nucleotides

The purines are not synthesised as free bases but are associated with ribose-5-phosphate

as complete nucleotides from the outset. Inosinic acid, which is formed initially, acts as

a precursor for the other purine nucleotides.

Pyrimidines have a similarly complex synthesis. The amino acids aspartate and glu-

tamine are involved in the synthesis of the precursor orotic acid. Note that unlike the

purines, the skeleton of pyrimidines is fully formed before association with the ribose-

5-phosphate moiety, which is itself derived from glucose (see biosynthesis of carbohy-

drates, above).

Ribonucleotides (as contained in RNA) are converted to deoxyribonucleotides (as

contained in DNA) by a reduction reaction that may involve vitamin B

acting as a

cofactor.

Biosynthesis of amino acids

A very limited number of microorganisms are able to utilise molecular nitrogen from

the atmosphere by incorporating it initially into ammonia and subsequently into or-

ganic compounds (see Chapter 7). Most organisms, however, need to have their ni-

trogen supplied as nitrate, nitrite, or ammonia itself. Ammonia can be incorporated

into organic nitrogen compounds in several ways, including glutamate formation from

α-ketoglutarate (see TCA cycle, above):

α-ketoglutarate + NH

+ NADPH* + H

−→ glutamate + NADP

+ H

(*Some species utilise NADH as their electron donor)

The amino group can subsequently be transferred from the glutamate to make other

amino acids by transamination reactions involving other keto acids:

e.g. glutamate + oxaloacetate −→ aspartate + α-ketoglutarate

Glutamate plays a central role in the biosynthesis of other amino acids, as it usually

donates the primary amino group of each:

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ANABOLIC REACTIONS 153

Glutamate

Pyruvic acid

C=O

COOH

COO

–

H-C-NH

COOH

Alanine

H-C-NH

COOH

α-Ketoglutarate

COO

–

C=O

COOH

According to the precursor molecule from which they derive, amino acids can be placed

into six ‘families’ (Figure 6.37). The precursors are intermediates in metabolic pathways

we have already encountered in this chapter, such as glycolysis or the TCA cycle. When

Pyruvate family

Pyruvate

3-Phospho-

glycerate

Alanine

Valine

Leucine

Serine Serine family

Aromatic family

Chorismate

Glycine

Cysteine

Phenylalanine

Tyrosine

Tryptophan

Phosphoenol-

pyruvate

Erythose 4-PO

Ribose 5-PO

GLYCOLYSISTCA CYCLE

Histidinol

Histidine

Lysine

Threonine

Isoleucine

Histidine family

Glutamate family

Glutamine

Arginine

Proline

Glutamate

Aspartate

Oxalo-

acetate

α-Keto-

glutarate

Aspartate

Aspartate family

Asparagine

PENTOSE PHOSPHATE

PATHWAY (see Fig.6.19)

Figure 6.37 Amino acid biosynthesis. The carbon skeleton of amino acids is obtained from

a limited number of precursor molecules, mainly intermediates in glycolysis or the TCA cycle.

The amino group originally derives from inorganic sources, but can then be transferred from

one organic molecule to another

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154 MICROBIAL METABOLISM

amino acids are broken down, they are likewise broken down into a handful of metabolic

intermediates, which then feed into the TCA cycle.

The regulation of metabolism

Microorganisms, like the rest of us, live in a changing world, and their needs do not

always remain the same. It would be highly inefﬁcient and (frequently wasteful) if all

their metabolic reactions were going on with equal intensity all the time, regardless of

whether they were needed. Over evolutionary time, regulation systems have developed,

so that metabolism is tailored to the prevailing conditions.

Essentially, this regulation involves controlling the activity of enzymes which direct

the many biochemical reactions occurring in each cell. This can be done by:

directly affecting enzyme activity, or

indirectly, at the genetic level, by controlling the level at which enzymes are

synthesised.

Direct control of enzymatic activity occurs by the mechanism of feedback inhibition

(see Box 6.5), whereby the ﬁnal product of a metabolic pathway acts as an inhibitor to

the enzyme that catalyses an early step (usually the ﬁrst) in the pathway. It thus prevents

Box 6.5 Feedback inhibition

Biosynthetic pathways exist as a series of enzyme-mediated reactions, leading to

a final product required by the cell for structural or metabolic purposes. But what

happens if for some reason, the consumption of the final product slows down, or

even stops? Feedback inhibition, also known, perhaps more descriptively, as ‘end-

product inhibition’, ensures that excess amounts of the end product are not synthe-

sised. The pathways leading to the synthesis of many amino acids are regulated

in this way, for example isoleucine, which is synthesised from another amino acid,

threonine, via a series of intermediates:

Threonine Isoleucine

Here, the isoleucine itself acts as an inhibitor of threonine deaminase, the enzyme

which starts off the pathway. It does this by binding to an allosteric site on the enzyme,

distorting it and preventing its active site from binding to threonine. Note, that by

inhibiting the early part of the pathway, we not only prevent further production of

isoleucine but also unnecessary breakdown of threonine. When levels of isoleucine

starts to run low, less will be available to block the threonine deaminase, and thus

the pathway starts to function again.

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TEST YOURSELF 155

more of its own formation. When the concentration of the product subsequently falls

below a certain level, it is no longer inhibitory, and biosynthesis resumes.

Regulation of metabolic pathways can also be achieved by controlling whether or

not an enzyme is synthesised in the ﬁrst place, and if so, the rate at which it is produced.

This is done at the DNA level, by one of two mechanisms, induction and repression,

which respectively ‘switch on’ and ‘switch off’ the machinery of protein synthesis dis-

cussed earlier in this chapter. These are discussed under the heading ‘Regulation of gene

expression’ in Chapter 11

Test yourself

1 Reactions which involve the breakdown of compounds are collectively

termed

and synthetic (building up) ones are termed .

2 Enzymes lower the

of a reaction.

3 The

of an enzyme is the part that is actually involved in

catalysis.

4 At pH values away from the optimum,

of ‘R’ groups causes denat-

uration of proteins.

5 When a molecule

an electron, it is said to be oxidised.

6 Compounds which are good electron donors have a strongly

redox

potential.

7 The energy ‘currency’ of cellular metabolism is

8 The ﬁrst step in glucose metabolism is

, which can occur with or

without

9 ATP may be formed from ADP by

phosphorylation,

phosphorylation or phosphorylation.

10 Before entering the TCA cycle, pyruvate must be converted to the two-carbon

compound

. This type of reaction, in which a molecule of carbon

dioxide is removed, is called a

11 The main product of the TCA cycle is reducing power, in the form of

and .

12 The role of oxygen in aerobic respiration is to act as the

in the electron transport chain.

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156 MICROBIAL METABOLISM

13 As envisaged by the chemiosmotic theory of Peter Mitchell, protons are

pumped out across a membrane, generating a

anaerobes are able to utilise anaerobic respiration if oxygen is

unavailable.

15 The two commonest products of fermentation reactions are

and

16 Fermentation yields far less ATP than aerobic respiration because it does not

involve an

17 Fatty acids are broken down

carbons at a time by the process of

are organisms that can derive their energy from the oxidation of

inorganic compounds such as ammonia or reduced iron.

19 Photosynthesis carried out by green and purple bacteria differs from that

carried out by algae and cyanobacteria in that no

is generated.

20 In the light reactions of photosynthesis, light energy and an electron donor

are used to generate

and .

21 In the dark reactions that make up the

cycle, carbon dioxide is

assimilated into glyceraldehyde-3-phosphate, which is then converted to glu-

cose in a series of steps that are the reverse of

22 Anoxygenic photosynthetic bacteria are unable to use

as an elec-

tron donor, and therefore use compounds such as hydrogen sulphide or suc-

cinate.

23 Organisms unable to synthesise glucose from inorganic carbon must derive

it from other organic compound by the process of

24 In amino acid synthesis, -NH

groups may be transferred between com-

pounds in

reactions.

25 The inhibition of a metabolic pathway by its ﬁnal product is termed end-

product or

inhibition.

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Part III

Microbial Diversity

157