Thymidylate, a vital DNA precursor,
is synthesized by thymidylate
synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially
widespread in anaerobes. TS encoded by thyX requires
a flavin adenine dinucleotide prosthetic group for activity. In the
oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine
5′-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate
(CH2THF), synthesizing 2′-deoxythymidine 5′-monophosphate
(dTMP). dTMP synthesis is a complex process, requiring the enzyme
to promote carbon transfer, probably by increasing the nucleophilicity
of dUMP and the electrophilicity of CH2THF, and reduction
of the transferred carbon. The mechanism of the oxidative half-reaction
was investigated by transient kinetics. Two intermediates were detected,
the first by a change in the flavin absorbance spectrum in stopped-flow
experiments and the second by the transient disappearance of deoxynucleotide
in acid quenching experiments. The effects of substrate analogues
and the behavior of mutated enzymes on these reactions lead to the
conclusion that activation of dUMP does not occur through a Michael-like
addition, the mechanism for the activation analogous with that of
the flavin-independent TS. Rather, we propose that the nucleophilicity
of dUMP is enhanced by electrostatic polarization upon binding to
the active site. This conclusion rationalizes many of our observations,
for instance, the markedly slower reactions when two arginine residues
that hydrogen bond with the uracil moiety of dUMP were mutated to
alanine. The activation of dUMP by polarization is consistent with
the majority of the published data on ThyX and provides a testable
mechanistic hypothesis.
Rib fractures result in serious morbidity and mortality after trauma. Although there is ongoing debate about surgical rib fixation, it is increasingly important for some patients. Minimally invasive techniques for rib fixation are gaining traction within the trauma community. We present an observational experience at our level 1 trauma center with our first 10 cases of video-assisted thoracoscopic surgery (VATS) internal rib fixation. Video-assisted thoracoscopic surgery internal plates are especially helpful for rib fractures under the scapula, which are difficult to access traditionally. This technique is also excellent at reducing complex segmental fractures as the bridge can span across multiple fractures with a single post on either side. They also work well for posterior fractures where multiple screws cannot be placed. Video-assisted thoracoscopic surgery internal rib fixation is a viable and exciting option for surgical fixation. The plates work particularly well for certain fracture patterns.
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