The Three-Dimensional Structural Basis of Type II Hyperprolinemia

Abstract

Type II hyperprolinemia is an autosomal recessive disorder caused by a deficiency in Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH, aka ALDH4A1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. Here we report the first structure of human P5CDH and investigate the impact of the hyperprolinemia-associated mutation of Ser352 to Leu on the structure and catalytic properties of the enzyme. The 2.5 Å resolution crystal structure of human P5CDH was determined using experimental phasing. Structures of the mutant enzymes S352A (2.4 Å) and S352L (2.85 Å) were determined to elucidate the structural consequences of altering Ser352. Structures of the 93%-identical mouse P5CDH complexed with sulfate ion (1.3 Å resolution), glutamate (1.5 Å), and NAD+ (1.5 Å) were determined to obtain high resolution views of the active site. Together, the structures show that Ser352 occupies a hydrophilic pocket and is connected via water-mediated hydrogen bonds to catalytic Cys348. Mutation of Ser352 to Leu is shown to abolish catalytic activity and eliminate NAD+ binding. Analysis of the S352A mutant shows that these functional defects are caused by the introduction of the nonpolar Leu352 side chain rather than the removal of the Ser352 hydroxyl. The S352L structure shows that the mutation induces a dramatic 8-Å rearrangement of the catalytic loop. Because of this conformational change, Ser349 is not positioned to interact with the aldehyde substrate, conserved Glu447 is no longer poised to bind NAD+, and Cys348 faces the wrong direction for nucleophilic attack. These structural alterations render the enzyme inactive.

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion of Trypanosoma cruzi

“…1A) and Ser 340 ) enzymes, in which the specific residues are likely to be involved in catalytic processes, as reported previously in archaea and human orthologs (Fig. 1A) (25,66).…”

“…DFT calculations also indicate that the imino-protonated cyclic form (P5C) is thermodynamically preferred over the zwitterionic form of ␥GS. Accordingly, crystallographic data indicate that ␥GS is the substrate of T. thermophilus ⌬ 1 -pyrroline-5-carboxylate dehydrogenase and that the nucleophilic attack of the thiol portion of Cys 336 to the ␥GS carbonyl yields an intermediate hemithioacetal, which is subsequently converted to glutamate via NAD ϩ -mediated hydrolysis (25,66). Assuming that the active site of the T. cruzi P5CDH is similar to that of T. thermophilus, the deprotonation of the -SH group of the cysteine residue (whose pK a is unknown but can be assumed to be ϳ6 -8) (81) might be crucial to the oxidation of ␥GS because the thiolate is a more efficient nucleophile than its corresponding protonated form.…”

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

“…Although the mechanism of monofunctional human P5CDH has been established (18,19), ours is the first such study for PutAs.…”

“…Stopped flow methods demonstrated that the oxidative half-reaction with CoQ 1 is rate-limiting in the PRODH reaction (17). Although the kinetic mechanism of the P5CDH domain has yet to be fully characterized in EcPutA, it likely follows an ordered ternary mechanism as described for human P5CDH (18,19).…”

Proline Mechanisms of Stress Survival

“…Next, PRODH transfers two electrons from reduced flavin to an electron acceptor such as ubiquinone in the inner membrane of the mitochondria (or cytoplasmic membrane in prokaryotes) (86,135). After P5C spontaneously converts into GSA, GSA is oxidized to L-glutamate by P5C dehydrogenase (P5CDH) using nicotinamide adenine dinucleotide as an electron receptor (6,121). Glutamate generated by proline oxidation enters the tricarboxylic acid cycle after enzymatic conversion to a-ketogluturate.…”