Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

170 Chapter 7
In the case of erythromycin the polyketide is assembled from the threecarbon
unit of propionyl CoA, which is carboxylated to give methylmalonyl
CoA. Acyl transfer and carbon–carbon bond formation takes place through
the a-carbon of a methylmalonyl-thioester, in the same way as is shown in
Figure 7.14, giving a-methyl-b-keto-thioesters at each stage. Each ‘module’
of enzyme activities contains the enzymes required for the assembly of a
new b-keto-thioester and its subsequent modiWcation. For example, the Wrst
module contains ketosynthase (KS) and acyltransferase (AT) activities to make
the new b-keto-thioester, and a ketoreductase (KR) activity to reduce the
b-keto-thioester to a b-R-hydroxy-thioester, and so on. Each of the multifunctional
polyketide synthases contains two such ‘modules’ of enzymatic activities.
At the end of the third polyketide synthase is a thioesterase (TE) activity
which catalyses the intramolecular lactonisation via a serine acyl enzyme intermediate.
Subsequent modiWcation of the polyketide precursor to erythromycin
A occurs by separate P 450 mono-oxygenase and glycosyl transferase enzymes.
7.5 Carboxylases: use of biotin
We have already seen examples of nucleophilic attack of a carbanion equivalent
onto aldehyde and ester electrophiles. There are Wnally a number of examples of
nucleophilic attack of carbanion equivalents onto carbon dioxide to generate
carboxylic acid products.
We have just seen that the carboxylation of acetyl CoA to give malonyl
CoA is an important step in fatty acid and polyketide natural product biosynthesis.
This step is catalysed by acetyl CoA carboxylase. This enzyme uses acetyl
CoA and bicarbonate as substrates, but also requires adenosine triphosphate
(ATP), which is converted to adenosine diphosphate (ADP) and inorganic
phosphate (P i ), and the cofactor biotin. Biotin was Wrst isolated from egg
yolk in 1936, and was found to act as a vitamin whose deWciency causes
dermatitis. Its structure is a bicyclic ring system containing a substituted urea
functional group which is involved in its catalytic function. The biotin
cofactor is covalently attached to the e-amino side chain of an active site lysine
residue.
How does such an apparently unreactive chemical structure serve to activate
carbon dioxide for these carboxylation reactions, and what is the role of ATP in
the reaction These questions were answered by a series of experiments with
isotopically labelled bicarbonate substrates. Bicarbonate is rapidly formed from
carbon dioxide in aqueous solution and is the substrate for biotin-dependent
carboxylases. Incubation of biotin-dependent b-methylcrotonyl-CoA carboxylase
with 14 C-bicarbonate and ATP gave an intermediate 14 C-labelled enzyme
species. Methylation with diazomethane followed by degradation of the
enzyme structure revealed that the 14 C-label was covalently attached to the biotin
cofactor, in the form of a carbon dioxide adduct onto N 1 of the cofactor. This