Progress in the Chemistry of Cytochalasans
93
were that, in addition to the in vivo experiments proving that MycB is related to
DA, in vitro enzymatic assays also gave direct evidence that MycB catalyzes the DA
cycloaddition with stereospecificity. Considering the similarity of the cyclization
patterns of these two types of compounds, the possibility of the existence of specific
DA enzymes in the cytochalasan pathway is high.
Additional evidence has come from a study of the pyrichalasin H (164) biosynthetic gene cluster conducted by Hantke and others [266] (Scheme 3D). Through
the knockout of pyiF in BGC, the accumulated intermediate metabolites B10–B17
all contain linear PKS moieties with several sites hydroxylated, and it could be
speculated that oxygenases downstream or outside the BGC lead to nonspecific
modifications to unstable intermediates. Furthermore, when ORF3, the homologous
enzyme of PyiF from another cryptic BGC, was reintroduced into the knockout
strain, the cyclic pyrichalasin H (164) was again produced. Although the in vivo
results obtained were quite convincing, it was unfortunate that attempts to obtain a
soluble PyiF protein failed. Direct evidence of a DAase may be expected from the
in vitro characterization of such an enzyme in the cytochalasan pathway, including
its control over the rate of cycloaddition and stereochemistry.
4.2.3 The Late Stage of Cytochalasan Biosynthesis
After the Diels–Alder cycloaddition, the cytochalasan biosynthesis pathway enters
the late stage, which involves a series of oxidative modifications. This could be
explained by the abundant oxidoreductases in the BGCs, including, flavin-dependent
oxidoreductase, cytochrome P450s, and other enzymes, with functions elucidated
continuously by in vivo and in vitro characterizations. In recent years, some novel
cytochalasans isolated by our group have been seen to undergo major changes in their
skeleton, mainly due to DA and nucleophilic addition reactions between cytochalasans and a polyketide small molecule. Their proposed biosynthesis also will be
covered in this Section.
The flavin-dependent monooxygenase (FMO) CcsB catalyzes two consecutive
oxidations to form a unique carbonate structure (Scheme 4A). Its function was
verified by in vivo experiments (gene knockout) and in vitro enzymatic assays by
Tang’s group [267]. Interestingly, CcsB catalyzes the first oxygen insertion through
a classical Baeyer–Villiger mechanism to form the ester intermediate C2, while the
second oxygen insertion can occur only when the substrate C1 has a vinylogous 1,5diketo system. Other FAD-dependent oxidoreductases (OXRs) are involved mainly
in ketone-alcohol conversions.
Another major group of oxidases in the cytochalasan BGCs are cytochrome P450s.
These activate C–H bonds at different sites of the molecules, forming various derivatives. Wan and others elucidated the functions of two P450 enzymes in pyi (the BGC
of pyrichalasin H) by gene knockout [8] (Scheme 4B, left). Realizing that these
P450s had substrate promiscuity, they applied a combinatorial biosynthesis strategy
to replace the pyi P450s with P450s from other homologous BGCs, thus obtaining
non-natural novel cytochalasans such as C4 [55] (Scheme 4B, right).
93
were that, in addition to the in vivo experiments proving that MycB is related to
DA, in vitro enzymatic assays also gave direct evidence that MycB catalyzes the DA
cycloaddition with stereospecificity. Considering the similarity of the cyclization
patterns of these two types of compounds, the possibility of the existence of specific
DA enzymes in the cytochalasan pathway is high.
Additional evidence has come from a study of the pyrichalasin H (164) biosynthetic gene cluster conducted by Hantke and others [266] (Scheme 3D). Through
the knockout of pyiF in BGC, the accumulated intermediate metabolites B10–B17
all contain linear PKS moieties with several sites hydroxylated, and it could be
speculated that oxygenases downstream or outside the BGC lead to nonspecific
modifications to unstable intermediates. Furthermore, when ORF3, the homologous
enzyme of PyiF from another cryptic BGC, was reintroduced into the knockout
strain, the cyclic pyrichalasin H (164) was again produced. Although the in vivo
results obtained were quite convincing, it was unfortunate that attempts to obtain a
soluble PyiF protein failed. Direct evidence of a DAase may be expected from the
in vitro characterization of such an enzyme in the cytochalasan pathway, including
its control over the rate of cycloaddition and stereochemistry.
4.2.3 The Late Stage of Cytochalasan Biosynthesis
After the Diels–Alder cycloaddition, the cytochalasan biosynthesis pathway enters
the late stage, which involves a series of oxidative modifications. This could be
explained by the abundant oxidoreductases in the BGCs, including, flavin-dependent
oxidoreductase, cytochrome P450s, and other enzymes, with functions elucidated
continuously by in vivo and in vitro characterizations. In recent years, some novel
cytochalasans isolated by our group have been seen to undergo major changes in their
skeleton, mainly due to DA and nucleophilic addition reactions between cytochalasans and a polyketide small molecule. Their proposed biosynthesis also will be
covered in this Section.
The flavin-dependent monooxygenase (FMO) CcsB catalyzes two consecutive
oxidations to form a unique carbonate structure (Scheme 4A). Its function was
verified by in vivo experiments (gene knockout) and in vitro enzymatic assays by
Tang’s group [267]. Interestingly, CcsB catalyzes the first oxygen insertion through
a classical Baeyer–Villiger mechanism to form the ester intermediate C2, while the
second oxygen insertion can occur only when the substrate C1 has a vinylogous 1,5diketo system. Other FAD-dependent oxidoreductases (OXRs) are involved mainly
in ketone-alcohol conversions.
Another major group of oxidases in the cytochalasan BGCs are cytochrome P450s.
These activate C–H bonds at different sites of the molecules, forming various derivatives. Wan and others elucidated the functions of two P450 enzymes in pyi (the BGC
of pyrichalasin H) by gene knockout [8] (Scheme 4B, left). Realizing that these
P450s had substrate promiscuity, they applied a combinatorial biosynthesis strategy
to replace the pyi P450s with P450s from other homologous BGCs, thus obtaining
non-natural novel cytochalasans such as C4 [55] (Scheme 4B, right).