5 research outputs found
Tuning the Production of Variable Length, Fluorescent Polyisoprenoids Using Surfactant-Controlled Enzymatic Synthesis
Bactoprenyl diphosphate (BPP), a
two-<i>E</i> eight-<i>Z</i> configuration C<sub>55</sub> isoprenoid, serves as a critical
anchor for the biosynthesis of complex glycans central to bacterial
survival and pathogenesis. BPP is formed by the polymerase undecaprenyl
pyrophosphate synthase (UppS), which catalyzes the elongation of a
single farnesyl diphosphate (FPP) with eight <i>Z-</i>configuration
isoprene units from eight isopentenyl diphosphates. <i>In vitro</i> analysis of UppS and other polyprenyl diphosphate synthases requires
the addition of a surfactant such as Triton X-100 to stimulate the
release of the hydrophobic product from the enzyme for effective and
efficient turnover. Here using a fluorescent 2-nitrileanilinogeranyl
diphosphate analogue of FPP, we have found that a wide range of surfactants
can stimulate release of product from UppS and that the structure
of the surfactant has a major impact on the lengths of products produced
by the protein. Of particular importance, shorter chain surfactants
promote the release of isoprenoids with four to six <i>Z</i>-configuration isoprene additions, while larger chain surfactants
promote the formation of natural isoprenoid lengths (8<i>Z</i>) and larger. We have found that the product chain lengths can be
readily controlled and coarsely tuned by adjusting surfactant identity,
concentration, and reaction time. We have also found that binary mixtures
of just two surfactants can be used to fine-tune isoprenoid lengths.
The surfactant effects discovered do not appear to be significantly
altered with an alternative isoprenoid substrate. However, the surfactant
effects do appear to be dependent on differences in UppS between bacterial
species. This work provides new insights into surfactant effects in
enzymology and highlights how these effects can be leveraged for the
chemoenzymatic synthesis of otherwise difficult to obtain glycan biosynthesis
probes. This work also provides key reagents for the systematic analysis
of structure–activity relationships between glycan biosynthesis
enzymes and isoprenoid structure