Graz University of Technology Austria Institute of Biochemistry ...

unusual amino acids, i.e. hydroxypyridylhomothreonine (HPHT) and aminohexuronic acid
with an N-glycosidically linked base:
Although the chemical structures of nikkomycins have been known since the 1970s, only a
few biosynthetic steps have been investigated in detail. The steps leading to the synthesis of
aminohexuronic acid are unclear. Originally, it was hypothesized that the aminohexuronic
acid moiety is generated by addition of an enolpyruvyl moiety from phosphoenolpyruvate
(PEP) to either the uridine or the 4-formyl-4-imidazolin-2-one analog at the 5’-position of
ribose. This step is then followed by rather speculative modifications to yield the
aminohexuronic acid precursor. In contrast to this hypothesis, we could recently demonstrate
that UMP rather than uridine serves as the acceptor for the enolpyruvyl moiety, a reaction
catalyzed by an enzyme encoded by a gene of the nikkomycin operon termed nikO.
Furthermore, we could demonstrate that it is attached to the 3’- rather than the 5’-position of
UMP. These results are very intriguing since none of the nikkomycins synthesized possess an
enolpyruvyl group in this position of the sugar moiety. Hence, it must be concluded that the
resulting 3’-enolpyruvyl-UMP is subject to rearrangement reactions where the enolpyruvyl is
detached from its 3’-position and transferred to the 5’-position of the ensuing aminohexuronic
acid moiety. Co-crystallization of NikO with fosfomycin yielded rod – like crystals diffracting
up to 2.5 Å. A synchrotron dataset was measured at the Swiss Light Source and the structure
was solved by molecular replacement using UDP-N-acetylglucosamine enolpyruvyl
transferase (PDB code: 2rl1) as a model. Two molecules were found in the asymmetric unit
exhibiting an inverse α,β-barrel fold with helices forming the tightly packed core and sheets
shielding the hydrophobic core from the solvent:
Eech chain is comprised of two inverse α,β-barrel subunits, which are connected by a hinge
region. The final structure was refined to final R/R free values of 17% and 19%, respectively.
Crystallization trials of NikO in the presence of its product, 3’-EP-UMP, are currently under
way.
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