Ligation events influence ALG-2 dimerization.

ALG-2 dimerization was studied using Förster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined, the sensitized emission from AF647 serves as a sensitive probe of dimer formation.

Structural Investigation of a Dimeric Variant of Pyruvate Kinase Muscle Isoform 2.

Pyruvate kinase muscle isoform 2 (PKM2) catalyzes the terminal step in glycolysis, transferring a phosphoryl group from phosphoenolpyruvate to ADP, to produce pyruvate and ATP. PKM2 activity is allosterically regulated by fructose 1,6-bisphosphate (FBP), an upstream glycolytic intermediate. FBP stabilizes the tetrameric form of the enzyme.

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.

Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis.

The bifunctional flavoenzyme proline utilization A (PutA) catalyzes the two-step oxidation of proline to glutamate using separate proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase active sites. Because PutAs catalyze sequential reactions, they are good systems for studying how metabolic enzymes communicate via substrate channeling. Although mechanistically similar, PutAs vary widely in domain architecture, oligomeric state, and quaternary structure, and these variations represent different structural solutions to the problem of sequestering a reactive metabolite.

Impact of disease-Linked mutations targeting the oligomerization interfaces of aldehyde dehydrogenase 7A1.

Aldehyde dehydrogenase 7A1 (ALDH7A1) is involved in lysine catabolism, catalyzing the oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Certain mutations in the ALDH7A1 gene, which are presumed to reduce catalytic activity, cause an autosomal recessive seizure disorder known as pyridoxine-dependent epilepsy (PDE). Although the genetic association between ALDH7A1 and PDE is well established, little is known about the impact of PDE-mutations on the structure and catalytic function of the enzyme.

EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt).

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.

Site-directed mutagenesis of rat α-parvalbumin: replacement of canonical CD-site residues with their non-consensus counterparts from rat β-parvalbumin.

Rat β-parvalbumin (β-PV) displays low divalent-ion affinity. Its CD site is distinguished by six non-consensus residues--the "CD-loop residues"--at positions 49, 50, 57-60. Additionally, leucine occupies position 85, rather than phenylalanine, the β-lineage-consensus residue.

Structural studies of yeast Δ(1)-pyrroline-5-carboxylate dehydrogenase (ALDH4A1): active site flexibility and oligomeric state.

The proline catabolic enzyme Δ(1)-pyrroline-5-carboxylate dehydrogenase (ALDH4A1) catalyzes the NAD(+)-dependent oxidation of γ-glutamate semialdehyde to l-glutamate. In Saccharomyces cerevisiae, ALDH4A1 is encoded by the PUT2 gene and known as Put2p. Here we report the steady-state kinetic parameters of the purified recombinant enzyme, two crystal structures of Put2p, and the determination of the oligomeric state and quaternary structure from small-angle X-ray scattering and sedimentation velocity.

Characterization of avian thymic hormone and chicken parvalbumin 3 target cells.

Avian thymic hormone (ATH) is a β-parvalbumin produced by epithelial cells in the thymic cortex and in the eyes of chickens. Chicken parvalbumin 3 (CPV3) is a homologous protein produced in the thymus and in hair cells of the chicken ear. ATH circulates in the blood on a five-day cycle and stimulates cell-mediated immunity when administered to young chickens.

Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein-ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca(2+) affinity and relatively low Mg(2+) affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and -enthalpies for both ions challenging.

Solution structures of polcalcin Phl p 7 in three ligation states: Apo-, hemi-Mg2+-bound, and fully Ca2+-bound.

Polcalcins are small EF-hand proteins believed to assist in regulating pollen-tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain-swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.

The three-dimensional structural basis of type II hyperprolinemia.

Type II hyperprolinemia is an autosomal recessive disorder caused by a deficiency in Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH; also known as ALDH4A1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. Here, we report the first structure of human P5CDH (HsP5CDH) and investigate the impact of the hyperprolinemia-associated mutation of Ser352 to Leu on the structure and catalytic properties of the enzyme. The 2.

Heightened stability of polcalcin Phl p 7 is correlated with strategic placement of apolar residues.

Phl p 7 exhibits atypical conformational stability and a diminutive denaturational heat capacity increment, ΔC(p). Because exposure of apolar surface largely dictates the magnitude of ΔC(p), a depressed value could signify an unusually compact unfolded state. The volume of the denatured state ensemble (DSE) is evidently inversely correlated with mean hydrophobicity [Pace et al.

Crystal structure and immunogenicity of the class C acid phosphatase from Pasteurella multocida.

Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP).

Solution structures of chicken parvalbumin 3 in the Ca(2+)-free and Ca(2+)-bound states.

Birds express two β-parvalbumin isoforms, parvalbumin 3 and avian thymic hormone (ATH). Parvalbumin 3 from chicken (CPV3) is identical to rat β-parvalbumin (β-PV) at 75 of 108 residues. CPV3 displays intermediate Ca(2+) affinity--higher than that of rat β-parvalbumin, but lower than that of ATH.

Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens.

The crystal structure of the enzyme phosphoglucomutase from Salmonella typhimurium (StPGM) is reported at 1.7 A resolution. This is the first high-resolution structural characterization of a bacterial protein from this large enzyme family, which has a central role in metabolism and is also important to bacterial virulence and infectivity.

Disparate impact of the S33V mutation on conformational stability in rat β-parvalbumin (oncomodulin) and chicken parvalbumin 3.

Rat β-parvalbumin (β-PV) and chicken parvalbumin 3 (CPV3) exhibit diminished Ca(2+) affinity. Their sequences, 70% identical, are unusual in that serine replaces the consensus residue, valine, at position 33. Reasoning that the substitution of a compact, polar hydroxymethyl moiety for a bulky, apolar isopropyl group might contribute to the attenuated Ca(2+) affinities, we have characterized the S33V variants of both proteins.

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA.

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane-embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N-terminal residues 2 through 11 of SecA and the C-terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation.

Structure of avian thymic hormone, a high-affinity avian beta-parvalbumin, in the Ca2+-free and Ca2+-bound states.

Originally isolated on the basis of its capacity to stimulate T-cell maturation and proliferation, avian thymic hormone (ATH) is nevertheless a parvalbumin, one of two beta-lineage isoforms expressed in birds. We recently learned that addition of Ca(2+)-free ATH to a solution of 8-anilinonaphthalene-1-sulfonate (ANS) markedly increases ANS emission. This behavior, not observed in the presence of Ca(2+), suggests that apolar surface area buried in the Ca(2+)-bound state becomes solvent accessible upon Ca(2+) removal.

Polcalcin divalent ion-binding behavior and thermal stability: comparison of Bet v 4, Bra n 1, and Bra n 2 to Phl p 7.

Polcalcins are pollen-specific proteins containing two EF-hands. Atypically, the C-terminal EF-hand binding loop in Phl p 7 (from timothy grass) harbors five, rather than four, anionic side chains, due to replacement of the consensus serine at -x by aspartate. This arrangement has been shown to heighten parvalbumin Ca(2+) affinity.

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum.

The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD(+)-dependent Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.

Conformational stabilities of guinea pig OCP1 and OCP2.

OCP1 and OCP2, the most abundant proteins in the cochlea, are evidently subunits of an SCF E3 ubiquitin ligase. Although transcribed from a distinct gene, OCP2 is identical to Skp1. OCP1 is equivalent to the F-box protein known as Fbs1, Fbx2, or NFB42 - previously shown to bind N-glycosylated proteins and believed to function in the retrieval and recycling of misfolded proteins.

Characterization of a unique class C acid phosphatase from Clostridium perfringens.

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported.