Places delta-1-pyrroline-5-carboxylate dehydrogenase (1P5CDH / ALDH4A1) as the second of four mitochondrial enzymes of the trans-4-hydroxy-L-proline degradation pathway. The paper's own focus is the terminal enzyme HOGA1, not ALDH4A1.

Human enzyme kinetics and human apo/mutant structures establish NAD+-dependent GSA oxidation and homodimeric assembly. Mouse ligand complexes provide the structural template for a model accommodating hydroxylated GSA; hydroxy-substrate activity is described as established pathway biology, not newly measured in this study.

The type II hyperprolinemia mutation S352L abolishes catalytic activity and eliminates NAD+ binding by rearranging the catalytic loop.

Human MPC1-MPC2 cryo-EM structures resolve the substrate translocation pathway at the heterodimer interface. Co-expression/purification and structural preparation target these two subunits; the study does not quantify or experimentally exclude native ALDH4A1 association.

Six cryo-EM structures of human MPC in IMS-open, occluded and matrix-facing states define an alternating-access transport cycle carried out by an MPC1-MPC2 heterodimer.

Human S352L and G521fs(+1) alleles fail to complement P5CDH-deficient yeast and have no detectable P5CDH activity; P16L remains functional and is considered polymorphic.

Human-cell co-immunoprecipitation, recombinant complex assays, mitochondrial gel filtration and BN-PAGE support association of ALDH4A1 with MPC1-MPC2; added ALDH4A1 enhances reconstituted pyruvate transport. The reported trimer model does not by itself establish a universal native 1:1:1 stoichiometry.

The effect is catalytically independent: the type II hyperprolinemia substitution S352L, which abolishes P5C dehydrogenase activity, stimulates reconstituted pyruvate transport as well as wild-type ALDH4A1, and tumour suppression is likewise enzyme-activity independent.

MPC1 and MPC2 together already transport pyruvate in the same proteoliposome assay without ALDH4A1; ALDH4A1 enhances that activity rather than being required for it.

Cryo-EM structures of the human MPC1-2 complex in IMS-open and inhibitor-bound matrix-open states; the transport channel is formed by TM1 and TM2 of MPC1 packing against TM2 and TM1 of MPC2.

Cloned human P5CDH cDNAs (563-residue protein) whose expression conferred P5CDH activity and growth on proline in a P5CDH-deficient yeast strain; describes the enzyme as a mitochondrial matrix NAD+-dependent dehydrogenase catalyzing the second step of proline degradation.