Orotidine 5'-monophosphate decarboxylase (OMPDC) is one of the most efficient enzyme systems studied, enhancing the decarboxylation of OMP to uridine 5'-monophosphate (UMP) by ca. 17 orders of magnitude, primarily by reducing the enthalpy of activation by ca. 28 kcal/mol. Despite a substantial reduction in activation enthalpy, OMPDC requires 15 kcal/mol of activation energy post-ES complex formation. This study investigates the physical basis of how thermal energy from solvent collisions is directed into the active site of enzyme to enable efficient thermal activation of the reaction. Comparative study of temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX) for WT and mutant forms of enzymes has recently been shown to uncover site specific protein networks for thermal energy transfer from solvent to enzyme active sites. In this study, we interrogate region-specific changes in the enthalpic barrier for local protein flexibility using a native OMPDC from Methanothermobacter thermautotrophicus (Mt-OMPDC) and a single site variant (Leu123Ala) that alters the activation enthalpy for catalytic turnover. The data obtained implicate four spatially resolved, thermally sensitive networks that originate at different protein/solvent interfaces and terminate at sites surrounding the substrate near the substrate phosphate-binding region (R203), the substrate- ribose binding region (K42), and a reaction enhancing loop5 (S127). These are proposed to act synergistically, transiently optimizing the position and electrostatics of the reactive carboxylate of the substrate to facilitate activated complex formation. The uncovered complexity of thermal activation networks in Mt-OMPDC distinguishes this enzyme from other members of the TIM barrel family previously investigated by TDHDX. The new findings extend the essential role of protein scaffold dynamics in orchestrating enzyme activity, with broad implications for the design of highly efficient biocatalysts.

The ongoing pandemic of COVID-19, caused by the infection of SARS-CoV-2, has generated significant harm to the world economy and taken numerous lives. This syndrome is characterized by an acute inflammatory response, mainly in the lungs and kidneys. Accumulated evidence suggests that exogenous heparin might contribute to the alleviation of COVID-19 severity through anticoagulant and various non-anticoagulant mechanisms, including heparanase inhibition, chemokine and cytokine neutralization, leukocyte trafficking interference, viral cellular-entry obstruction, and extracellular cytotoxic histone neutralization. However, the side effects of heparin and potential drawbacks of administering heparin therapy need to be considered. Here, the current heparin therapy drawbacks were covered in great detail: structure-activity relationship (SAR) mystery, potential contamination, and anticoagulant activity. Considering these unfavorable effects, specific non-anticoagulant heparin derivatives with antiviral activity could be promising candidates to treat COVID-19. Furthermore, a structurally diverse library of non-anticoagulant heparin derivatives, constructed by chemical modification and enzymatic depolymerization, would contribute to a deeper understanding of SAR mystery. In short, targeting non-anticoagulant mechanisms may produce better therapeutic effects, overcoming the side effects in patients suffering from COVID-19 and other inflammatory disorders.

CYP3A5 is the primary CYP3A subfamily enzyme expressed in the human kidney and its aberrant expression may contribute to a broad spectrum of renal disorders. Pharmacogenetic studies have reported inconsistent linkages between CYP3A5 expression and hypertension, however, most investigators have considered CYP3A5*1 as active and CYP3A5*3 as an inactive allele. Observations of gender specific differences in CYP3A5*3/*3 protein expression suggest additional complexity in gene regulation that may underpin an environmentally responsive role for CYP3A5 in renal function. Reconciliation of the molecular mechanism driving conditional restoration of functional CYP3A5*3 expression from alternatively spliced transcripts, and validation of a morpholino-based approach for selectively suppressing renal CYP3A5 expression, is the focus of this work. Morpholinos targeting a cryptic splice acceptor created by the CYP3A5*3 mutation in intron 3 rescued functional CYP3A5 expression in vitro, and salt-sensitive cellular mechanisms regulating splicing and conditional expression of CYP3A5*3 transcripts are reported. The potential for a G-quadruplex (G4) in intron 3 to mediate restored splicing to exon 4 in CYP3A5*3 transcripts was also investigated. Finally, a proximal tubule microphysiological system (PT-MPS) was used to evaluate the safety profile of morpholinos in proximal tubule epithelial cells, highlighting their potential as a therapeutic platform for the treatment of renal disease.