Diethylaminobenzaldehyde Is a Covalent, Irreversible Inactivator of ALDH7A1

There is growing interest in aldehyde dehydrogenases (ALDHs) because of their overexpression in cancer stem cells and the ability to mediate resistance to cancer drugs. Here, we report the first crystal structure of an aldehyde dehydrogenase complexed with the inhibitor 4-diethylaminobenzaldehyde (DEAB). Contrary to the widely held belief that DEAB is a reversible inhibitor of ALDHs, we show that DEAB irreversibly inactivates ALDH7A1 via formation of a stable, covalent acyl-enzyme species

Crystal Structures and Kinetics of Monofunctional Proline Dehydrogenase Provide Insight into Substrate Recognition and Conformational Changes Associated with Flavin Reduction and Product Release

Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of proline to Δ¹-pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from <i>Deinococcus radiodurans</i> in the oxidized state complexed with the proline analogue l-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analogue binds against the <i>si</i> face of the FAD isoalloxazine and is protected from bulk solvent by helix α8 and the β1−α1 loop. The FAD ribityl chain adopts two conformations in the E–S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of <i>Deinococcus</i> PRODH. Reduction of the crystalline enzyme–inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1−α1 loop, and rupture of the Arg291–Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold, which suggests that flexibility of the β1−α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E–S complex suggest that conformational selection plays a role in substrate recognition

Steric Control of the Rate-Limiting Step of UDP-Galactopyranose Mutase

Galactose is an abundant monosaccharide found exclusively in mammals as galactopyranose (Gal<i>p</i>), the six-membered ring form of this sugar. In contrast, galactose appears in many pathogenic microorganisms as the five-membered ring form, galactofuranose (Gal<i>f</i>). Gal<i>f</i> biosynthesis begins with the conversion of UDP-Gal<i>p</i> to UDP-Gal<i>f</i> catalyzed by the flavoenzyme UDP-galactopyranose mutase (UGM). Because UGM is essential for the survival and proliferation of several pathogens, there is interest in understanding the catalytic mechanism to aid inhibitor development. Herein, we have used kinetic measurements and molecular dynamics simulations to explore the features of UGM that control the rate-limiting step (RLS). We show that UGM from the pathogenic fungus <i>Aspergillus fumigatus</i> also catalyzes the isomerization of UDP-arabinopyranose (UDP-Ara<i>p</i>), which differs from UDP-Gal<i>p</i> by lacking a -CH₂-OH substituent at the C5 position of the hexose ring. Unexpectedly, the RLS changed from a chemical step for the natural substrate to product release with UDP-Ara<i>p</i>. This result implicated residues that contact the -CH₂-OH of UDP-Gal<i>p</i> in controlling the mechanistic path. The mutation of one of these residues, Trp315, to Ala changed the RLS of the natural substrate to product release, similar to the wild-type enzyme with UDP-Ara<i>p</i>. Molecular dynamics simulations suggest that steric complementarity in the Michaelis complex is responsible for this distinct behavior. These results provide new insight into the UGM mechanism and, more generally, how steric factors in the enzyme active site control the free energy barriers along the reaction path

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

<i>Escherichia coli</i> proline utilization A (<i>Ec</i>PutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. <i>Ec</i>PutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of <i>Ec</i>PutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes <i>Ec</i>PutA to dissociate from the <i>put</i> regulon and bind to the cellular membrane. Major structure/function domains of <i>Ec</i>PutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for <i>Ec</i>PutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in <i>Ec</i>PutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs <i>Ec</i>PutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for <i>Ec</i>PutA functional switching

Contributions of Unique Active Site Residues of Eukaryotic UDP-Galactopyranose Mutases to Substrate Recognition and Active Site Dynamics

UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDP-galactofuranose. Absent in humans, galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. For these reasons, UGMs are targets for drug discovery. Here, we report a mutagenesis and structural study of the UGMs from Aspergillus fumigatus and Trypanosoma cruzi focused on active site residues that are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs. Kinetic analysis of the variants F66A, Y104A, Q107A, N207A, and Y317A (A. fumigatus numbering) show decreases in <i>k</i>_cat/<i>K</i>_M values of 200–1000-fold for the mutase reaction. In contrast, none of the mutations significantly affect the kinetics of enzyme activation by NADPH. These results indicate that the targeted residues are important for promoting the transition state conformation for UDP-galactofuranose formation. Crystal structures of the A. fumigatus mutant enzymes were determined in the presence and absence of UDP to understand the structural consequences of the mutations. The structures suggest important roles for Asn207 in stabilizing the closed active site, and Tyr317 in positioning of the uridine ring. Phe66 and the corresponding residue in Mycobacterium tuberculosis UGM (His68) play a role as the backstop, stabilizing the galactopyranose group for nucleophilic attack. Together, these results provide insight into the essentiality of the targeted residues for realizing maximal catalytic activity and a proposal for how conformational changes that close the active site are temporally related and coupled together

Identification of the NAD(P)H Binding Site of Eukaryotic UDP-Galactopyranose Mutase

UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD­(P)­H. Kinetic protein crystallography was used to obtain structures of oxidized <i>Aspergillus fumigatus</i> UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP⁺. NAD­(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, and thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All 12 residues that contact NADP­(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP­(H) binding sites. The structures address the longstanding question of how UGM binds NAD­(P)H and provide new opportunities for drug discovery

Importance of the C‑Terminus of Aldehyde Dehydrogenase 7A1 for Oligomerization and Catalytic Activity

Aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes the terminal step of lysine catabolism, the NAD⁺-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Structures of ALDH7A1 reveal the C-terminus is a gate that opens and closes in response to the binding of α-aminoadipate. In the closed state, the C-terminus of one protomer stabilizes the active site of the neighboring protomer in the dimer-of-dimers tetramer. Specifically, Ala505 and Gln506 interact with the conserved aldehyde anchor loop structure in the closed state. The apparent involvement of these residues in catalysis is significant because they are replaced by Pro505 and Lys506 in a genetic deletion (c.1512delG) that causes pyridoxine-dependent epilepsy. Inspired by the c.1512delG defect, we generated variant proteins harboring either A505P, Q506K, or both mutations (A505P/Q506K). Additionally, a C-terminal truncation mutant lacking the last eight residues was prepared. The catalytic behaviors of the variants were examined in steady-state kinetic assays, and their quaternary structures were examined by analytical ultracentrifugation. The mutant enzymes exhibit a profound kinetic defect characterized by markedly elevated Michaelis constants for α-aminoadipate semialdehyde, suggesting that the mutated residues are important for substrate binding. Furthermore, analyses of the in-solution oligomeric states revealed that the mutant enzymes are defective in tetramer formation. Overall, these results suggest that the C-terminus of ALDH7A1 is crucial for the maintenance of both the oligomeric state and the catalytic activity

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor–Electrophile Conjugate

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor–electrophile conjugate <b>5a</b> caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10² M^–1 min^–1. Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) <i>Biochemistry</i> <i>44</i>, 7704–7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor–electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121