Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.

Deposition of amyloid-β (Aβ) in Alzheimer’s disease (AD) is strongly correlated with the APOE genotype. However, the role of apolipoprotein E (apoE) in Aβ aggregation has remained unclear. Here we have used different apoE preparations, such as recombinant protein or protein isolated from cultured astrocytes, to examine the effect of apoE on the aggregation of both Aβ1–40 and Aβ1–42. The kinetics of aggregation, measured by the loss of fluorescence of tetramethylrhodamine-labeled Aβ, is shown to be dramatically slowed by the presence of substoichiometric concentrations of apoE. Using these concentrations, we conclude that apoE binds primarily to and affects the growth of oligomers that lead to the nuclei required for fibril growth. At higher apoE concentrations, the protein also binds to Aβ fibrils, resulting in fibril stabilization and a slower rate of fibril growth. The aggregation of Aβ1–40 is dependent on the apoE isoform, being the most dramatic for apoE4 and less so for apoE3 and apoE2. Our results indicate that the detrimental role of apoE4 in AD could be related to apoE-induced stabilization of the soluble but cytotoxic oligomeric forms and intermediates of Aβ, as well as fibril stabilization.

The apolipoprotein apoE plays a key role in cholesterol and lipid metabolism. There are three isoforms of this protein, one of which, apoE4, is the major risk factor for Alzheimer's Disease. At μM concentrations all lipid-free apoE isoforms exist primarily as monomers, dimers and tetramers. However, the molecular weight form of apoE that binds to lipid has not been clearly defined. We have examined the role of self-association of apoE with respect to interactions with phospholipids. Measurements of the time dependence of turbidity clearance of small unilamellar vesicles of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) upon addition of apoE show that higher molecular weight oligomers bind poorly if at all. The kinetic data can be described by a reaction model in which tetramers and dimers of apoE must first dissociate to monomers which then bind to the liposome surface in a fast and reversible manner. A slow but not readily reversible conformational conversion of the monomer then occurs. Prior knowledge of the rate constants for the association-dissociation process allows us to determine the rate constant of the conformational conversion. This rate constant is isoform dependent and appears to correlate with the stability of the apoE isoforms with the rate of dissociation of the apoE oligomers to monomers being the rate limiting process for lipidation. Differences in the lipidation kinetics between the apoE isoforms arise from their differences in the self-association behavior leading to the conclusion that selfassociation behavior may influence biological functions of apoE in an isoform dependent manner.Apoliprotein E (apoE) is a constituent of several plasma lipoproteins and plays a key role in the metabolism of cholesterol and triglycerides. There are three isoforms of the protein (apoE2, apoE3 and apoE4) that differ only by single amino acid changes. These isoforms differ markedly in their preferences for lipoprotein particles in the plasma and in their receptor binding abilities (1-3). In brief, apoE2 and apoE3 bind preferentially to high density lipoprotein (HDL) particles whereas apoE4 shows high affinity for very low density lipoprotein particles (VLDLs) (4). Additionally, apoE3 and apoE4 bind to low density lipoprotein receptors (LDLR) with high affinity but apoE2 binds only weakly (1-3). Importantly, apoE4 is associated with higher risk for Alzheimer's disease and for cardiovascular disease while apoE2 is associated with hyperlipoproteinamia (3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In spite of the profound differences in the outcomes of these diseases, the differences in the molecular properties of the apoE isoforms are still unclear.The complete structure of lipid-free wild-type ApoE is unknown but the ApoE monomer consists of two domains, an N-terminal domain (residue 1-191) and a C-terminal domain (residue 221-299) (18). The single amino acid changes occur in the N-terminal domain with apoE2 containing two cysteines (C112/C158), apoE4 with arginines replacing the two cysteines (R11...

The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1-261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1-90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nM, which is slightly higher than that of the PutA-DNA complex (K d ϳ 45 nM). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbonhelix-helix DNA binding motif in residues 1-47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nM for the PutA47-DNA complex. Thus, Nterminal residues 1-47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors.