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Enzymatic Carbon–Carbon Bond Formation 183
Pentalenene synthase F L D D F L D
Trichodiene synthase V L D D S K D
Aristolochene synthase L I D D V L E
Casbene synthase
L I D D T I D
Limonene synthase
V I D D I Y D
Farnesyl PP synthase V Q D D I L D
Geranylgeranyl PP synthase I A D D Y H N
Consensus
D D X X(D)
O
H
N
H
O
O
O −
N
H
O
− O
H
O
Mg 2+
− O
O −
R O P O P O −
O O
Figure 7.35 Mg 2þ -pyrophosphate binding motif.
How do these cyclase enzymes control the precise regiochemistry and
stereochemistry of these multi-step cyclisations In 1997 the crystal structure
of pentalenene synthase was solved. The active site consists of a conical hydrophobic
cleft, Xanked by six a-helices, lined with aromatic and non-polar residues.
The DDXXD motif is located near the upper edge of the cavity, as shown
in Figure 7.36. The identity of the catalytic base has not yet been identiWed, but
replacement of Phe-76 or Phe-77 by alanine gave at least a 10-fold reduction in
activity, suggesting that they may stabilise carbocationic intermediates through
-cation interactions. The presence of aromatic residues in key positions of the
wall of terpene cyclase active sites suggests that the -cation interaction, shown
in Figure 7.37, may have an important role in the speciWc stabilisation of the
carbocation intermediates.
Carbon–carbon bond formation via radical
intermediates
In Chapter 6 we have seen how redox enzymes are able to generate radical
intermediates in enzyme-catalysed reactions. In certain instances these
radical intermediates are used for carbon–carbon bond formation reactions.
The two examples which we shall examine are phenol radical couplings used in
natural product biosynthesis and in lignin biosynthesis.