C O M M U N I C A T I O N S
Table 1. Kinetic Parameters of MdpB2 towards Aromatic
Substrates Featuring 1,2-Hydroxy Acid Functionality (highlighted in
red)
Substrate
KM (µM)
kcat (min-1
)
rel kcat/KM
6
4
8
9
10
11
0.041 ( 0.005
2.1 ( 0.3
0.23 ( 0.05
1.2 ( 0.2
2.32 ( 0.06
0.59 ( 0.02
0.90 ( 0.03
0.86 ( 0.04
g0.0042a
1
0.0049
0.069
0.013
g0.0013a
a Values represent specific activities at 100 µM substrate
concentration.
MdpB2 can also utilize salicylic acid (8) and its 3-methyl derivative
(9), in addition to 4, as a substrate, albeit with 15-, 80-, and 200-
fold lower specificities than 6, respectively (Figure S2). In contrast,
MdpB2 barely recognized benzoic acid (10) and its methyl
derivative (11) as substrates, exhibiting specific activities of only
0.25 and 0.08 h-1, respectively, at substrate concentrations of 100
µM. Although the kinetic analyses were based on the ATP-
[32P]pyrophosphate exchange assays,9 formation of the correspond-
ing CoA thioester product was confirmed in all cases by HPLC
(Figure S3) and HRMS.9 In contrast to other aryl-CoA ligases such
as NcsB27 or SsfL1,13 MdpB2 is apparently more specific for the
1,2-hydroxy acid functionality, reminiscent of that reported for the
NcsB1 O-methyltransferase.6
Figure 2. HPLC analysis of MdpB2- and MdpB1-catalyzed sequential
conversion of 6 (O) to 5 ([): (A) MdpB2-catalyzed conversion of 6 (O) to
7 (b) (I) and control without enzyme (II) and (B) authentic standards of 7
(b) and 4 (3) (I) and time course of MdpB1-catalyzed conversion of 7 (b)
to 5 ([) and its degradation product 4 (3) upon 1.5 h (II) and 4.5 h (III)
incubation.
We next overproduced MdpB2 in E. coli and purified it to
homogeneity (Figure S1) to examine the predicted CoA ligase
activity in vitro. Upon incubation of 6 with MdpB2 in the presence
of ATP and CoA, a new product was detected by HPLC analysis
(Figure 2A) and identified as the corresponding CoA thioester 7.9
This suggested that MdpB2 may act, before MdpB1, on 6 as
depicted in Figure 1D.
In summary, this study characterizes a novel C-methyltransferase
that acts on a CoA-tethered aromatic substrate in natural product
biosynthesis. Relatively few C-methyltransferases act on aromatic
substrates in natural product biosynthesis.11 Notably, polyketomycin
(POK) also possesses the 4 moiety (Figure S4A) and the POK
biosynthetic gene cluster predicts proteins with high sequence
identities (65-73%) to MdpB (PokM1), MdpB1 (PokMT1), and
MdpB2 (PokM3) (Figure S4B).11a Indeed, a ∆pokMT1 strain
produced a 6-containing POK analogue, confirming PokMT1 as a
C-methyltransferase (Figure S4A). Our present in vitro work using
purified MdpB1 sets the stage to further characterize this unusual
enzyme capable of C-methylating a CoA-tethered aromatic sub-
strate. To our knowledge, only an O-methyltransferase acting on
caffeoyl-CoA has been described to date.12 Although the signifi-
cance of the unusual biosynthetic logic is unclear, MdpB1 may
represent an emerging class of C-methyltransferases exploitable for
tailoring CoA thioester-tethered intermediates in natural product
biosynthetic pathways.
We then chemically synthesized 49 and compared the steady-
state kinetic parameters of MdpB2 toward 4 (Figure 1C) and 6
(Figure 1D) to differentiate the two pathways. CoA ligase catalysis
occurs in two steps: (i) ATP-dependent activation of carboxylic
acids as acyl-AMPs and (ii) addition of CoA to the acyl-AMP to
generate the acyl-CoA product. Although the rate-limiting step of
the MdpB2 reaction is unknown, we probed adenylation specificity
using the ATP-[32P]pyrophosphate exchange assay, which has been
widely used for characterizing the substrate specificities of adeny-
lating enzymes.10 Although 4 was also converted to its CoA
thioester 5, whose identity was verified by HPLC analysis (Figure
S3A) and high resolution mass spectrometry (HRMS),9 MdpB2
apparently prefers 6 (kcat of 2.3 min-1 and KM of 0.041 µM) over
4 (kcat of 0.59 min-1 and KM of 2.1 µM) (Table 1 and Figure S2).
The 200-fold higher specificity (kcat/KM) of MdpB2 for 6 over 4
supports the proposed pathway in Figure 1D, implying that MdpB1
C-methylates the CoA activated 7 rather than the free acid 6 as a
substrate.
Acknowledgment. We thank the Analytical Instrumentation
Center of the School of Pharmacy, University of WisconsinsMadison
for support in obtaining NMR and mass spectrometric data. This
work was supported in part by NIH Grants CA78747 and
CA113297. J.L. is the recipient of a China Scholarship Council
fellowship, G.P.H. is the recipient of an NSERC (Canada) post-
doctoral fellowship, and Y.L. is the recipient of a Visiting Scholar
Fellowship from the Chinese Academy of Sciences.
The kinetic insight from MdpB2 prompted re-evaluation of
MdpB1 in light of the pathway proposed in Figure 1D. Incubation
of MdpB1 with 7 and SAM yielded two products in a time-
dependent fashion upon HPLC analysis (Figure 2B). One was the
expected product 5,9 and the other product was identified as 4,
indicating that 5 was not stable under the assay conditions. These
results corroborate the revised pathway of Figure 1D and highlight
its unusual biosynthetic logic of CoA-activation one step earlier
than chemically necessary and demonstrate that MdpB1 is a
C-methyltransferase specific for a CoA-tethered aromatic substrate.
Finally we characterized MdpB2 substrate promiscuity and
observed a requirement for the ortho-hydroxy and carboxylate
groups of the aromatic acid substrate. As summarized in Table 1,
Supporting Information Available: Full experimental details, SDS-
PAGE gels, kinetic data, HPLC chromatograms, and comparison
between MDP and POK biosynthesis. This material is available free
9
J. AM. CHEM. SOC. VOL. 132, NO. 36, 2010 12535