Y. Ding, M. Wang, J. Li et al.
Biochemical and Biophysical Research Communications 543 (2021) 8e14
3.3. Pau17 can catalyze the 6-hydroxylation of 2,3-DHBA
an unprecedented 3-HAA 6-hydroxylase. The product of Pau17
catalyzed reaction, 3,6-DHAA, may undergo further C-6 glycosy-
lated and C-4 hyoxylation to shape the special ring A structure of
PAUs. Those biosynthetic steps are currently being actively inves-
tigated in our laboratory.
The substrate promiscuity of Pau17 was then tested using 3-
amino-2-hydroxybenzoic acid, 3-hydroxybenzoic acid, and 2,3-
DHBA to substitute 3-HAA. Although the former two could not be
recognized by Pau17, the last candidate, 2,3-DHBA, was oxidized by
Pau17 to generate 2,3,6-trihydroxy benzoic acid (2,3,6-THBA),
which was identified by HR-MS analysis (m/z 169.0138 [MþH]þ,
calcd 169.0142) and a HPLC co-injection with authentic 2,3,6-THBA
(Fig. 3B). The 2,3-DHBA 6-hydroyxlation activity of Pau17 showed
that this enzyme has certain substrate promiscuity towards 3-HAA
analogues. In addition, the conversion of 2,3-DHBA to 2,3,6-THB
provided further evidence to support that Pau17 acts as a 3-HAA
6-hydroxylase in PAU biosynthesis.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported in part by the National Key R&D Pro-
gram of China (2018YFA0901900), Center for Ocean Mega-Science,
CAS (KEXUE2019GZ05), and the National Natural Science Founda-
tion of China (31670032 and 32025002).
4. Discussion
As a group of antibiotics with considerable inhibition activity
against tumor cell lines and varied gram-positive bacteria discov-
ered in 1960s (named as proceomycin or U-43,120 originally)
[3,22], PAUs were intensively studied for their potential use in the
treatment of infection diseases [23,24]. PAUs exhibited attracting
activities against some gram-positive bacteria pathogens including
multiple-antibiotic-resistant Staphylococcus aureus [23]. They could
also kill the S. aureus surviving within human or bovine poly-
morphonuclear leukocytes efficiently [24]. The biosynthetic studies
of PAUs were initiated by the feeding experiments of various pre-
cursors, which suggested that the fatty acyl chains connected to
paulomycose 7ʹʹ-OH of PAU A and B are derived from the degra-
dation products of isoleucine and valine, respectively [25]. Some
important progresses on understanding PAU biosynthesis have
been made after the pau biosynthetic gene clusters were assigned.
In the case of the special ring A moiety of PAUs, it was proposed that
Pau18 (Plm15), Pau19 (Plm16), and Pau20 (Plm17) can convert
chorismate to 3-HAA, an identified intermediate of PAU biosyn-
thesis [4,7,9]. We showed that Pau17 could convert 3-HAA and 2,3-
DHBA to RA-318 and 2,3,6-THBA, respectively. Both of the two
enzymatic assays and the in vivo results supported that Pau17 is a 3-
HAA 6-hydroxylase involved in PAU biosynthesis. In the PAUs
producers, the product of Pau17, 3,6-DHAA, may be taken by the
following biosynthetic enzymes immediately to avoid the attacks
from cellular nucleophiles and auto-oxidation.
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13