Structural and biochemical basis for the pathogenic mutations of methylmalonate semialdehyde dehydrogenase ALDH6A1

Methylmalonate semialdehyde dehydrogenase (ALDH6A1), encoded by the ALDH6A1 gene, is essential for the metabolic degradation of valine and thymine. Genetic alterations in ALDH6A1 lead to methylmalonate semialdehyde dehydrogenase deficiency (MMSDD), a rare disease with only five reported disease mutations, focusing on its molecular foundation but lacking in-depth mechanistic investigations. Therefore, the structural and biochemical properties of the ALDH6A1 mutants have not yet been thoroughly examined. Wild-type (WT) and mutant ALDH6A1 were constructed as plasmids and purified after prokaryotic expression to obtain conformationally homogeneous and pure protein. The structures of ALDH6A1 mutants (P62S, Y172H & R535C, S262Y, P421S, and G446R) were solved using cryo-electron microscopy. Based on the results of ALDH6A1 WT, enzyme activity and thermal stability experiments of their mutants were performed to explore the ALDH6A1’s biochemical characteristics. This study presents a structural analysis of the ALDH6A1 mutants, P62S, Y172H & R535C, S262Y, P421S, and G446R at resolutions of 3.70, 2.92, 3.12, 3.47, and 3.00 Å, respectively. However, the electronic density of ALDH6A1 P421S is poor, and it is difficult to fit into this density. Furthermore, the root-mean-square deviation (r.m.s.d.) of ALDH6A1 WT with these mutants was significant. This study revealed a tetrameric structure with closely interacting monomers, except for ALDH6A1 P62S, which forms a dimer, and is consistent with the principles of the aldehyde dehydrogenase family. Moreover, these disease mutations also affect enzyme activity and thermal stability. Our findings shed light on disease mutations that contribute to the properties of ALDH6A1 and lead to the genesis of MMSDD from structural and biochemical perspectives, which holds promise as a potential theoretical basis for this rare disease.