Selective oxygenation of proline derivatives
1387
during the initial screening (Fig. 2) and a confirmed
Acknowledgments
decrease in the substrate, the reaction products could not
be detected using HPLC or MS analysis. Recently,
2-methylthioproline was reported to be hydrolytically
unstable in aqueous solutions.20) The authors suggested
that the ampholytic nature of 2-methylthioproline affects
its analysis with HPLC and MS. Our difficulty in detect-
ing oxygenated products of L-thioproline was likely
attributable to the same phenomena. However, the appar-
ent decrease in substrate and formation of succinate were
clearly confirmed by comparison with the enzyme-free
control, and thus, the enzymes likely recognized
L-thioproline as a substrate.
We would like to thank Kyowa Hakko Bio Co. Ltd.
for kindly providing us with Hyp and HPA and for
helpful discussions.
Funding
This work was financially supported by the Adaptable and
Seamless Technology Transfer Program through target-driven
R&D, JST (AS231157E).
We revealed the substrate specificities of various pro-
line cis-hydroxylases toward proline derivatives. The
reactions and the corresponding products are summa-
rized in Fig. 3. Analysis of the reactivity to 15 proline
derivatives suggests that proline cis-hydroxylases rec-
ognized α-imino carboxylic acid moiety with an L-con-
figuration. To our knowledge, this is the first report on
the enzymatic synthesis of 2,3-cis-3,4-cis-3,4-dihy-
droxy-L-proline from cis-3-L-Hyp by direct oxygena-
tion. In nature, several 3,4-dihydroxyproline isomers
can be isolated from diatomic cell walls,21) toxic
peptides from mushrooms,22) and animal adhesive pro-
teins.23) Because of their sugar-like structures, glycosi-
dase inhibitors have been studied for their potential in
biological and therapeutic applications.24,25) For
instance, 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline shows
potent and competitive inhibition of α-galactosidase.26)
Furthermore, the organic synthesis of various 3,4-di-
hydroxyproline isomers has been reported from easily
available carbohydrates (e.g., pentose sugars)27–32) and
non-carbohydrates.7) However, these methods still have
limitations for industrial applications because complex
reaction steps and multiple purification procedures
result in an overall low yield.
Among four proline cis-hydroxylases, only CaPH
oxygenated 2-AZC. Although the detailed mechanism
of this specific recognition would be elucidated by
enzyme–substrate complex analysis, it was revealed
that CaPH lacks “Pro_3_hydrox_C” domain, which is a
C-terminal region conserved in other cis-hydroxylases,
by conserved domain search. This property would be
considered as one of the reasons for the ability to
oxygenate 2-AZC by CaPH.
In this paper, we demonstrated that proline
cis-hydroxylases possess relaxed substrate specificity,
and they have the potential to efficiently produce
oxygenated proline derivatives in a one-step reaction.
Furthermore, proline cis-hydroxylases are a class of
2-oxoglutarate-dependent dioxygenases that do not
require expensive NAD(P)H or redox partner proteins.
From an industrial point of view, 2-oxoglutarate is
relatively cheap and is supplied from the more reason-
ably priced glucose through the metabolic pathway.
Indeed, trans-4-L-Hyp and cis-3-L-Hyp can be
produced by E. coli fermentation processes suggesting
that the enzymes in this study have the capacity for
adaptation to industrial use for oxygenation of various
proline derivatives.
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