Binding versus Enzymatic Processing of ε-Trimethyllysine Dioxygenase Substrate Analogues

Abstract

ε-Trimethyllysine dioxygenase (TMLD) is a non-heme Fe(II) and α-ketoglutarate dependent oxygenase that catalyzes the stereospecific hydroxylation of ε-trimethyl- l -lysine (TML) to β-hydroxy-TML during the first step of l -carnitine biosynthesis. Targeting TMLD with inhibitors is a viable strategy for the treatment of cardiovascular diseases. Herein, we report a methodology for isothermal titration calorimetry analysis of TMLD substrate analogue binding to the enzyme. Despite the high structural similarity of the tested compounds, two different binding mechanisms (enthalpy- and entropy-driven) were observed, giving insight into the ligand (substrate) selectivity of TMLD. We demonstrate that the method allows distinguishing a natural substrate-like binding mode, which correlates with the ability of the compounds to serve as substrates in the TMLD catalytic reaction.

Structural and Functional Studies of Proteins Involved in the Regulation of Fatty Acid Metabolism

“…Overall, it can be seen that the active site of MBP-TMLD is not blocked by other metal ions and effective substrate binding occurs only in the presence of both cofactors Fe(II)/αKG or their isosteres. The obtained data were described in the author's publication [34]. A. MBP-TMLD in complex with OGA (2-fold excess) titrated with TML.…”

“…2.9). The synthetic procedures were described in the author's publication [34]. Despite the variability in the chain length, all D-isomers [D-TML (9), δ-trimethyl-Dornithine (10), and ζ-trimethyl-D-homolysine (11)], as well as the ε-trimethylaminohexanoate (12) did not interact with MBP-TMLD.…”

“…Comparison of novel substrates of TMLD [34] with known substrates of BBOX [ Modelling data reveal that compounds 1, 4, and 6 have a highly similar position in the active site (e.g., Fig. 2.11 A) with no penalties for the bioactive conformation.…”

“…Such compounds may be used for the de novo TMLD inhibitor design due to longer residence times. The obtained data were described in the author's publication [34]. All figures of the ITC and enzymatic reaction experiments are available in the supplementary material of the publication.…”