Production and characterization of homologous protoporphyrinogen IX oxidase (PPO) proteins: Evidence that small N-terminal amino acid changes do not impact protein function.

Transgenic soybean, cotton, and maize tolerant to protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides have been developed by introduction of a bacterial-derived PPO targeted into the chloroplast. PPO is a membrane-associated protein with an intrinsic tendency for aggregation, making expression, purification, and formulation at high concentrations difficult. In this study, transgenic PPO expressed in three crops was demonstrated to exhibit up to a 13 amino acid sequence difference in the N-terminus due to differential processing of the chloroplast transit peptide (CTP).

Biochemical, structural, and computational analyses of two new clinically identified missense mutations of ALDH7A1.

Aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes a step of lysine catabolism. Certain missense mutations in the ALDH7A1 gene cause pyridoxine dependent epilepsy (PDE), a rare autosomal neurometabolic disorder with recessive inheritance that affects almost 1:65,000 live births and is classically characterized by recurrent seizures from the neonatal period. We report a biochemical, structural, and computational study of two novel ALDH7A1 missense mutations that were identified in a child with rare recurrent seizures from the third month of life.

Structural and functional analysis of two SHMT8 variants associated with soybean cyst nematode resistance.

Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5-phosphate-dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN-resistant soybean cultivar (cv.

Regulation of AUXIN RESPONSE FACTOR condensation and nucleo-cytoplasmic partitioning.

Auxin critically regulates plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARF protein condensation attenuates ARF activity, resulting in dramatic shifts in the auxin transcriptional landscape.

Probing the function of a ligand-modulated dynamic tunnel in bifunctional proline utilization A (PutA).

In many bacteria, the reactions of proline catabolism are catalyzed by the bifunctional enzyme known as proline utilization A (PutA). PutA catalyzes the two-step oxidation of l-proline to l-glutamate using distinct proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites, which are separated by over 40 Å and connected by a complex tunnel system. The tunnel system consists of a main tunnel that connects the two active sites and functions in substrate channeling, plus six ancillary tunnels whose functions are unknown.

Structural and Biochemical Characterization of the Flavin-Dependent Siderophore-Interacting Protein from .

is an opportunistic pathogen with a high mortality rate due to multi-drug-resistant strains. The synthesis and uptake of the iron-chelating siderophores acinetobactin (Acb) and preacinetobactin (pre-Acb) have been shown to be essential for virulence. Here, we report the kinetic and structural characterization of BauF, a flavin-dependent siderophore-interacting protein (SIP) required for the reduction of Fe(III) bound to Acb/pre-Acb and release of Fe(II).

Impact of missense mutations in the ALDH7A1 gene on enzyme structure and catalytic function.

Certain mutations in the ALDH7A1 gene cause pyridoxine-dependent epilepsy (PDE), an autosomal recessive metabolic disease characterized by seizures, and in some cases, intellectual disability. The mutational spectrum of PDE is vast and includes over 70 missense mutations. This review summarizes the current state of biochemical and biophysical research on the impact of PDE missense mutations on the structure and catalytic activity of ALDH7A1.

Inhibition, crystal structures, and in-solution oligomeric structure of aldehyde dehydrogenase 9A1.

Aldehyde dehydrogenase 9A1 (ALDH9A1) is a human enzyme that catalyzes the NAD-dependent oxidation of the carnitine precursor 4-trimethylaminobutyraldehyde to 4-N-trimethylaminobutyrate. Here we show that the broad-spectrum ALDH inhibitor diethylaminobenzaldehyde (DEAB) reversibly inhibits ALDH9A1 in a time-dependent manner. Possible mechanisms of inhibition include covalent reversible inactivation involving the thiohemiacetal intermediate and slow, tight-binding inhibition.

Impaired folate binding of serine hydroxymethyltransferase 8 from soybean underlies resistance to the soybean cyst nematode.

Management of the agricultural pathogen soybean cyst nematode (SCN) relies on the use of SCN-resistant soybean cultivars, a strategy that has been failing in recent years. An underutilized source of resistance in the soybean genotype Peking is linked to two polymorphisms in serine hydroxy-methyltransferase 8 (SHMT8). SHMT is a pyridoxal 5'-phosphate-dependent enzyme that converts l-serine and (6)-tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate.

Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine-dependent epilepsy.

Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxine-dependent epilepsy (PDE). Missense mutations of Glu427, especially Glu427Gln, account for ~30% of the mutated alleles in PDE patients, and thus Glu427 has been referred to as a mutation hot spot of PDE. Glu427 is invariant in the ALDH superfamily and forms ionic hydrogen bonds with the nicotinamide ribose of the NAD cofactor.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness.

Structural and biochemical consequences of pyridoxine-dependent epilepsy mutations that target the aldehyde binding site of aldehyde dehydrogenase ALDH7A1.

In humans, certain mutations in the gene encoding aldehyde dehydrogenase 7A1 are associated with pyridoxine-dependent epilepsy (PDE). Understanding the impact of PDE-causing mutations on the structure and activity of ALDH7A1 could allow for the prediction of symptom-severity and aid the development of patient-specific medical treatments. Herein, we investigate the biochemical and structural consequences of PDE missense mutations targeting residues in the aldehyde substrate binding site: N167S, P169S, A171V, G174V, and W175G.

Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants.

Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD-dependent glutamate γ-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate γ-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate.

NAD promotes assembly of the active tetramer of aldehyde dehydrogenase 7A1.

Nicotinamide adenine dinucleotide (NAD) is the redox cofactor of many enzymes, including the vast aldehyde dehydrogenase (ALDH) superfamily. Although the function of NAD(H) in hydride transfer is established, its influence on protein structure is less understood. Herein, we show that NAD -binding promotes assembly of the ALDH7A1 tetramer.

Redox Modulation of Oligomeric State in Proline Utilization A.

Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure.

Determination of protein oligomeric structure from small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer.

Structural Basis for the Substrate Inhibition of Proline Utilization A by Proline.

Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of l-proline to l-glutamate using spatially separated proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.

Flavin-N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme.

The flavin-dependent enzyme 2-haloacrylate hydratase (2-HAH) catalyzes the conversion of 2-chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2-HAH was shown to be monomeric in solution and contained a non-covalent, yet tightly bound, flavin adenine dinucleotide (FAD).

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.

Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1.

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ-pyrroline-5-carboxylate (P5C) to proline. Mutations in the gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target.

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.

Refining the nuclear auxin response pathway through structural biology.

Auxin is a key regulator of plant growth and development. Classical molecular and genetic techniques employed over the past 20 years identified the major players in auxin-mediated gene expression and suggest a canonical auxin response pathway. In recent years, structural and biophysical studies clarified the molecular details of auxin perception, the recognition of DNA by auxin transcription factors, and the interaction of auxin transcription factors with repressor proteins.

Defining a two-pronged structural model for PB1 (Phox/Bem1p) domain interaction in plant auxin responses.

Phox/Bem1p (PB1) domains are universal structural modules that use surfaces of different charge for protein-protein association. In plants, PB1-mediated interactions of auxin response factors (ARF) and auxin/indole 3-acetic acid inducible proteins regulate transcriptional events modulated by the phytohormone auxin. Here we investigate the thermodynamic and structural basis for Arabidopsis thaliana ARF7 PB1 domain self-interaction.