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Appendix 4 281
(4) Reverse of normal biotin mechanism. Attack of N-1 of biotin cofactor onto
oxaloacetate gives pyruvate and carboxy-biotin intermediate, which carboxylates
propionyl CoA to give methylmalonyl CoA.
(5) Attack of TPP anion onto keto group gives tetrahedral adduct. Cleavage of
a,b-bond using TPP as electron sink gives enamine intermediate. This
reacts with aldehyde of second substrate to give another tetrahedral adduct.
Detachment from cofactor regenerates TPP anion.
(6) Attack of TPP anion onto ketone gives tetrahedral adduct. Decarboxylation
gives enamine intermediate as in normal mechanism. At this point the
enamine intermediate is oxidised by FAD to give acetyl adduct (probably
via H transfer followed by single electron transfer). Hydrolysis by attack of
water gives acetate product, and regenerates TPP anion. FADH 2 reoxidised
to FAD by O 2 .
(7) 1,3-migration of pyrophosphate to give linalyl PP. Attack at C-1 to form
six-membered ring gives tertiary carbonium ion. Formation of second ring
with concerted attack of pyrophosphate gives product. No positional isotope
exchange in pyrophosphate means that pyrophosphate is bound very
tightly (to Mg 2þ ) by enzyme, and is not even free to rotate.
OPP
OPP
− OPP
(8) Reaction to copalyl PP commenced by protonation of terminal alkene to
give tertiary carbonium ion. Two ring closures followed by loss of proton to
form C5C. Loss of pyrophosphate followed by formation of six-membered
ring. Closure to form Wnal Wve-membered ring followed by 1,2-alkyl shift
and elimination.
OPP
H
OPP
OPP
H
H
H
+
H
H +
H
H
H
(9) Aromatic precursor made from tetraketide intermediate which is methylated
by SAM: carbanion formation between Wrst and second ketones is
followed by attack on terminal thioester carbonyl. Formation of phenoxy
radical para to methyl group. Carbon–carbon bond formation via radical
coupling. Re-aromatisation of left-hand ring followed by attack of phenoxide
onto ab-unsaturated ketone of right-hand ring.