Ricerc@Sapienza

Carnitine, a crucial metabolite in eukariotes energy metabolism, is responsible for the transport of acyl and acetyl activated groups into the mitochondrial matrix, where their oxidation takes place. Mammals obtain carnitine primarily from diet, although they are able to synthesise it. The carnitine biosinthesis pathway has been well-characterized in invertebrates, yeasts and mammals. In mammals, the genes encoding three out of the four enzymes involved in the pathway have been identified and their enzyme products characterized. The identity of the remaining enzyme, which has been dubbed 3-hydroxy-N6-trimethyllysine aldolase (HTMLA) and catalyses the retroaldol cleavage of HTML (a ß-hydroxyamino acid) into glycyne and 4-N-trimethylaminobutyraldehyde (TMABA), remains a mystery.

It has been proposed that HTMLA coincides with cytosolic serine hydroxymethyltransferase (SHMT1), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that also exists as a mitochondrial isoform (SHMT2). However, this hypothesis has never been verified. Other PLP-dependent enzymes may have the capability to catalyse HTML cleavage. Threonine aldolases, for instance, are good candidates. Notably, although both mouse and human genomes possess a threonine aldolase gene, in humans this gene is not expressed.

The goal of this project is to identify the enzyme that plays the role of HTMLA in humans. A computational screening of all human PLP-dependent enzymes, including SHMT1 and SHMT2, will evaluate their potential capability to bind HTML and catalyse its retroaldol cleavage into glycine and TMABA. Human SHMT1, SHMT2, and any other PLP-dependent enzyme selected in the computational screening, will be characterized in vitro with respect to their actual capability to cleave HTML into glycine and TMABA. In vivo studies on cancer cell lines, in which SHMT1 and SHMT2 expression will be knockdown, will establish whether these enzymes are actually involved in the carnitine biosynthesis pathway.