Chemistry Reference
In-Depth Information
converted first to 3-methylglutaconyl-ACP then to 3-methylcrotonyl-ACP, which
is the gained intermediate for subsequent formation of the cyclopropyl ring.
Insights into the mechanism of the CurF ECH
2
decarboxylation have been gained
based on the recent crystal structure of the curacin ECH
2
domain (64). Addi-
tional
in vitro
evidence for the function of these enzymes has been generated
using proteins from the PksX pathway of
Bacillus subtilis
(46) and the myx-
ovirescin pathway from
Myxococcus xanthus
(58). Prior to the identification
of bacillaene as the product of the PksX pathway, Calderone et al. (46) and
Dorrestein et al. (57) reported the function of several discrete enzymes. Using
radioactive biochemical assays together with mass spectrometry, they assigned
functional roles to AcpK, PksC, the tandem ACPs in PksL, PksF, PksG, PksH,
and PksI. Using the model acceptor ACP, acetoacetyl-ACP, and malonyl-CoA in
combination with the above proteins, a
2
-isoprenyl-
S-
carrier protein was gen-
erated (46). Most recently, a similar
in vitro
investigation was conducted using
the homologous enzymes from the myxovirescin pathway (58). The HMGS cas-
sette reaction sequence proposed above held fast for the myxovirescin pathway,
although the generation of the propionyl- or methylmalonyl-
S
-ACP could not be
demonstrated. The authors have suggested that perhaps additional enzymes are
yet to be identified to fill these roles to complete the
β
-ethylation at C16 in
10
.
Two variations on the HMGS cassette theme already have been identified. In the
biosynthesis of bryostatin
2
, and leinamycin
8
, one or both of the ECH-mediated
steps is likely omitted based on the final natural product structures. The details
of these deviations have not yet been established.
Recently,
in vivo
evidence for the function of these HMGS cassettes has
come from the M uller lab (49, 50, 65). To date, all HMGS cassette proteins
for myxovirescin A (TaB/TaC, TaE/TaF, TaK, TaX, TaY) has been individually
deleted, and the impact on the products of the engineered
Myxococcus xanthus
strains has been analyzed. In addition, analysis of products from
taV (the
trans-acting AT),
taH (a cytochrome P450 that is thought to hydroxylate
the HMGS-installed
taQ (an
O
-methyl
transferase necessary for completing the transformation to the final methoxy
methyl functionality) strains have provided insights into this complex pathway.
Production of myxovirescin A was abolished (or greatly reduced) in all above
deletion strains. Appearance of novel myxovirescin analogs (
β
-methyl group at C12 of
10
), and
β
-methyl vs
β
-ethyl
at C16) in the
taF
strains
appears to be a result of TaB or TaC
complementation, which provides direct evidence for TaE/TaF in the formation
of the ethyl branch point. However, independent biochemical verification of TaF
function has been difficult to obtain (58).
taE &
7.3.4 HMGS Cassette Architecture
Analysis of the placement of the known HMGS cassettes identified to date into
their biosynthetic clusters reveals a variety of possible architectures (Fig. 7.6).
For example, the ECH
2
decarboxylase exists as a discrete enzyme downstream
of the ECH
1
dehydratase (mupirocin and others), as an N-terminal domain of
