10.1002/anie.202010042
Angewandte Chemie International Edition
COMMUNICATION
kinase (Adk)[11] was introduced into a scaled-up SfaB-catalyzed
reaction that was initiated by a relatively reduced ATP input (acid :
amine : ATP (10 mM) = 1 : 1 : 0.1). The results showed that the
yield of an example amide 6 was increased by ca. 49% with
inclusion of the Adk-Ppt system, demonstrating the practicability
of utilizing a recycling system to reduce the demand of cofactor
(Figure S41-43, yield of 6 without/with Adk-Ppt system =
24.7/36.8 mg, 8.6/12.8%, 36 h).
The authors declare no conflict of interest.
Keywords: biocatalysis • adenylation • amide synthetase•
substrate promiscuity • natural product
Reference
To further investigate whether the amidation and thioesterification
of acyl-AMPs were enzymatic or non-enzymatic processes in this
work, the necessity of SfaB catalysis for amide and thioester
formation needs to be confirmed. For this purpose, the
corresponding acyl-AMP and the partner amino acid or SNAC
should be added to the reaction with or without the presence of
SfaB for analysis. However, it was difficult for us to separate acyl-
AMPs due to its instability during lyophilization or organic
extraction. Hence, a size-exclusion column (10 KDa) was
introduced to remove the his-tagged SfaB (ca. 59.8 KDa) in the
reaction mixture after the acid being adenylated, and the
additional SfaB and nucleaphile were supplymented to the filtered
reaction system in the subsequent time-course assay (Figure 5A).
The biosyntheses of two amides (6 and 7) and one thioester (16)
were examined (Figure 5B). When the nucleaphile (L-arg or
SNAC) was added to the flowthrough at a concentration of 60 mM,
the time-course analyses revealed that the reaction velocities of
the SfaB-supplymented samples (red filled circles) were
significantly increased by 620 (6), 619 (7), and 11 (16) times
compared to those of the SfaB-free ones (red hollow circles),
respectively. The results were similar in the other experimental
set with the nucleaphiles at a concentration of 6 mM (blue circles).
Therefore, it is conclusively demonstrated that the amidation and
thioesterification in this study were completed by both enzymatic
and spontaneous nucleophilic attack, and the enzyme catalysis
played a dominant role.
In summary, SfaB is an adenylate-forming enzyme that
adenylates 3-isocyanobutanoic acid for the biosynthesis of the
diisonitrile natural product SF2768 in Streptomyces, and the
byproducts discovered accidentally in vitro reminded us that this
enzyme can directly catalyze the formation of amide bonds as an
amide synthetase. The considerable substrate flexibility allows us
to enzymatically synthesize diverse nonnative N-fatty acyl amides
and fatty acyl-CoA/SNAC thioesters with SfaB in vitro. It is also
demonstrated that the inclusion of an established ATP recycling
system for reducing the consumption of ATP in the SfaB-
catalyzed reaction is practically feasible. In the future, the yield
could be increased by means of directed evolution or whole-cell
catalysis or other appropriate methods, thereby making SfaB a
promising candidate for biocatalysis.
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Acknowledgements
This work is supported by the National Natural Science
Foundation of China (31870089), the Natural Science Foundation
for Distinguished Young Scholars of Hubei Province of China (No.
2018CFA069), and the Fundamental Research Funds for the
Central Universities (No. 2662018PY053). We thank Xing Zhang
(School of Chemistry, Sun Yat-sen University) for his assistance
with NMR experiments.
Conflict of interest
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