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Abstract The vacuolar sorting receptors (VSRs) are specific to plants and are responsible for sorting and transporting particular proteins from the trans-Golgi network to the vacuole. This process is critically important for various cellular functions, including storing nutrients during seed development. Despite many years of intense studies on VSRs, a complete relation between function and structure has not yet been revealed. Here, we present the crystal structure of the entire luminal region of glycosylated VSR1 from Arabidopsis thaliana (AtVSR1) for the first time. The structure provides insights into the tertiary and quaternary structures of VSR1, which are composed of an N-terminal protease-associated (PA) domain, a unique central region, and one epidermal growth factor (EGF)-like domain followed by two disordered EGF-like domains. The structure of VSR1 exhibits unique characteristics, the significance of which is yet to be fully understood.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI’s unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Flavonoids are potent antioxidants that play a role in defense against pathogens, UV-radiation, and the detoxification of reactive oxygen species. Dihydroflavonol 4-reductase (DFR) and flavanone 4-reductase (FNR) reduce dihydroflavonols and flavanones, respectively, using NAD(P)H to produce flavan-(3)-4-(di)ols in flavonoid biosynthesis. Anthocyanidin reductase (ANR) reduces anthocyanidins to flavan-3-ols. In addition to their sequences, the 3D structures of recombinant DFR, FNR and ANR from sorghum and switchgrass showed a high level of similarity. The catalytic mechanism, substrate-specificity and key residues of three reductases were deduced from crystal structures, site-directed mutagenesis, molecular docking, kinetics, and thermodynamic ana-lyses. Although DFR displayed its highest activity against dihydroflavonols, it also showed activity against flavanones and anthocyanidins. It was inhibited by the flavonol quercetin and high concentrations of dihydroflavonols/flavonones. SbFNR1 and SbFNR2 did not show any activity against dihydroflavonols. However, SbFNR1 displayed activity against flavanones and ANR activity against two anthocyanidins, cyanidin and pelargonidin. Therefore, SbFNR1 and SbFNR2 could be specific ANR isozymes without delphinidin activity. Sorghum has high concentrations of 3-deoxyanthocyanidins in vivo, supporting the observed high activity of SbDFR against flavonols. Mining of expression data indicated substantial induction of these three reductase genes in both switchgrass and sorghum in response to biotic stress. Key signature sequences for proper DFR/ANR classification are proposed and could form the basis for future metabolic engineering of flavonoid metabolism.

Cytochrome P450 reductase (CPR) is a multi-domain protein that acts as a redox partner of cytochrome P450s. The CPR contains a flavin adenine dinucleotide (FAD)–binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting domain. To achieve catalytic events, the FMN-binding domain needs to move relative to the FAD-binding domain, and this high flexibility complicates structural determination in high-resolution by X-ray crystallography. Here, we demonstrate a seeding technique of sorghum CPR crystals for resolution improvement, which can be applied to other poorly diffracting protein crystals. Protein expression is completed using an E. coli cell line with a high protein yield and purified using chromatography techniques. Crystals are screened using an automated 96-well plating robot. Poorly diffracting crystals are originally grown using a hanging drop method from successful trials observed in sitting drops. A macro seeding technique is applied by transferring crystal clusters to fresh conditions without nucleation to increase crystal size. Prior to diffraction, a dehydration technique is applied by serial transfer to higher precipitant concentrations. Thus, an increase in resolution by 7 Å is achieved by limiting the inopportune effects of the flexibility inherent to the domains of CPR, and secondary structures of SbCPR2c are observed.Graphical abstract:

APX is a key antioxidant enzyme in higher plants, scavenging H2O2 with ascorbate in several cellular compartments. Here, we report the crystal structures of cytosolic ascorbate peroxidase from switchgrass (Panicum virgatum L., Pvi), a strategic feedstock plant with several end uses. The overall structure of PviAPX was similar to the structures of other APX family members, with a bound ascorbate molecule at the ɣ-heme edge pocket as in other APXs. Our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Significantly, our study suggested that PviAPX can oxidize a broad range of phenylpropanoids with δ-meso site in a rather similar efficiency, which reflects its role in the fortification of cell walls in response to insect feeding. Based on detailed structural and kinetic analyses and molecular docking, as well as that of closely related APX enzymes, the critical residues in each substrate-binding site of PviAPX are proposed. Taken together, these observations shed new light on the function and catalysis of PviAPX, and potentially benefit efforts improve plant health and biomass quality in bioenergy and forage crops.

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamases (ESBLs) are commonly associated with Gram-negative, hospital-acquired infections worldwide. Several beta-lactamase inhibitors, such as clavulanate, are used to inhibit the activity of these enzymes. To understand the mechanism of CTX-M-15 activity, we have determined the crystal structures of CTX-M-15 in complex with two specific classes of beta-lactam compounds, desfuroylceftiofur (DFC) and ampicillin, and an inhibitor, clavulanic acid. The crystal structures revealed that Ser70 and five other residues (Lys73, Tyr105, Glu166, Ser130, and Ser237) participate in catalysis and binding of those compounds. Based on analysis of steady-state kinetics, thermodynamic data, and molecular docking to both wild-type and S70A mutant structures, we determined that CTX-M-15 has a similar affinity for all beta-lactam compounds (ceftiofur, nitrocefin, DFC, and ampicillin), but with lower affinity for clavulanic acid. A catalytic mechanism for tested β-lactams and two-step inhibition mechanism of clavulanic acid were proposed. CTX-M-15 showed a higher activity toward DFC and nitrocefin, but significantly lower activity toward ampicillin and ceftiofur. The interaction between CTX-M-15 and both ampicillin and ceftiofur displayed a higher entropic but lower enthalpic effect, compared with DFC and nitrocefin. DFC, a metabolite of ceftiofur, displayed lower entropy and higher enthalpy than ceftiofur. This finding suggests that compounds containing amine moiety (e.g., ampicillin) and the furfural moiety (e.g., ceftiofur) could hinder the hydrolytic activity of CTX-M-15.

NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV0) of the opening transition. While the ΔV0 characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.

The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal–chelate complexes. Microorganisms can transport free EDTA, but not metal–EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure–function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal–chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.

PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 μM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol.

Mycobacterium tuberculosis, the pathogen responsible for tuberculosis (TB), is the leading cause of death from infectious disease worldwide. The class A serine β-lactamase BlaC confers Mycobacterium tuberculosis resistance to conventional β-lactam antibiotics. As the primary mechanism of bacterial resistance to β-lactam antibiotics, the expression of a β-lactamase by Mycobacterium tuberculosis results in hydrolysis of the β-lactam ring and deactivation of these antibiotics. In this study, we conducted protein X-ray crystallographic analysis of the inactivation of BlaC, upon exposure to the inhibitor bis(benzoyl) phosphate. Crystal structure data confirms that serine β-lactamase is phosphorylated at the catalytic serine residue (Ser-70) by this phosphate-based inactivator. This new crystallographic evidence suggests a mechanism for phosphorylation of BlaC inhibition by bis(benzoyl) phosphate over acylation. Additionally, we confirmed that bis(benzoyl) phosphate inactivated BlaC in a time-dependent manner.

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O2 and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2−AOS−AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.

Calsequestrin (CASQ) is a major Ca2+-storage/buffer protein present in the sarcoplasmic reticulum of both skeletal (CASQ1) and cardiac (CASQ2) muscles. CASQ has significant affinity for a number of pharmaceutical drugs with known muscular toxicities. Our approach, with in silico molecular docking, single crystal X-ray diffraction, and isothermal titration calorimetry (ITC), identified three distinct binding pockets on the surface of CASQ2, which overlap with 2-methyl-2,4-pentanediol (MPD) binding sites observed in the crystal structure. Those three receptor sites based on canine CASQ1 crystal structure gave a high correlation (R2 = 0.80) to our ITC data. Daunomycin, doxorubicin, thioridazine, and trifluoperazine showed strong affinity to the S1 site, which is a central cavity formed between three domains of CASQ2. Some of the moderate-affinity drugs and some high-affinity drugs like amlodipine and verapamil displayed their binding into S2 sites, which are the thioredoxin-like fold present in each CASQ domain. Docking predictions combined with dissociation constants imply that presence of large aromatic cores and less flexible functional groups determines the strength of binding affinity to CASQ. In addition, the predicted binding pockets for both caffeine and epigallocatechin overlapped with the S1 and S2 sites, suggesting competitive inhibition by these natural compounds as a plausible explanation for their antagonistic effects on cardiotoxic side effects.

2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.

The association between altruistic values, religious values and pro-social behaviour is well documented, though mainly in North America and across disparate demographic groups. However, we currently have no data that focus on the relationships between personal values, religious values and pro-social behaviour across many different countries. Our study provides this data. We surveyed the values and pro-social behaviour (giving donations and volunteering) of university students in 14 different countries, thus achieving a unique cross-national perspective. We also included questions about materialistic values, which have hitherto been largely assumed to cause a reduction in pro-social behaviour. Our findings show that altruistic and religious values are positively significant in explaining variations in pro-social behaviour, but that materialistic values are not negatively correlated with pro-social behaviour. Our study thus suggests that, in the modern world, materialistic, religious and altruistic values can combine in complex ways to determine pro-social behaviour and that this combination varies across countries and cultures. In the discussion section, we draw conclusions that also relate to religious institutions and their diaconia.

Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc2Man1), while cardiac calsequestrin had five additional mannoses (GlcNAc2Man6). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca2+-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic “guiderail”-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca2+ concentrations, and proper glycosylation, in turn, guarantees both effective Ca2+ storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.

Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of calsequestrin as [Ca2+] in the SR-measured with a calsequestrin-fused biosensor-was lowered. We found that calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca2+] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric calsequestrin network and catastrophic structural changes in the SR. [ABSTRACT FROM AUTHOR]

Leaf senescence is the terminal stage in the development of perennial plants. Massive physiological changes occur that lead to the shut down of photosynthesis and a cessation of growth. Leaf senescence involves the selective destruction of the chloroplast as the site of photosynthesis. Here, we show that 13-lipoxygenase (13-LOX) accomplishes a key role in the destruction of chloroplasts in senescing plants and propose a critical role of its NH2-terminal chloroplast transit peptide. The 13-LOX enzyme identified here accumulated in the plastid envelope and catalyzed the dioxygenation of unsaturated membrane fatty acids, leading to a selective destruction of the chloroplast and the release of stromal constituents. Because 13-LOX pathway products comprise compounds involved in insect deterrence and pathogen defense (volatile aldehydes and oxylipins), a mechanism of unmolested nitrogen and carbon relocation is suggested that occurs from leaves to seeds and roots during fall. [ABSTRACT FROM AUTHOR]

PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 µM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol. [ABSTRACT FROM AUTHOR]

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor, SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midribll mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low Km for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion. [ABSTRACT FROM AUTHOR]

Hydroxycinnamoyltransferase (HCT) from sorghum (Sorghum bicolor) participates in an early step of the phenylpropanoid pathway, exchanging coenzyme A (CoA) esterified to p-coumaric acid with shikimic or quinic acid as intermediates in the biosynthesis of the monolignols coniferyl alcohol and sinapyl alcohol. In order to elucidate the mode of action of this enzyme, we have determined the crystal structures of SbHCT in its apo-form and ternary complex with shikimate and p-coumaroyl-CoA, which was converted to its product during crystal soaking. The structure revealed the roles of threonine-36, serine-38, tyrosine-40, histidine-162, arginine-371, and threonine-384 in catalysis and specificity. Based on the exact chemistry of p-coumaroyl-CoA and shikimic acid in the active site and an analysis of kinetic and thermodynamic data of the wild type and mutants, we propose a role for histidine-162 and threonine-36 in the catalytic mechanism of HCT. Considering the calorimetric data, substrate binding of SbHCT should occur sequentially, with p-coumaroyl-CoA binding prior to the acyl acceptor molecule. While some HCTs can use both shikimate and quinate as an acyl acceptor, SbHCT displays low activity toward quinate. Comparison of the structure of sorghum HCT with the HCT involved in chlorogenic acid synthesis in coffee (Coffea canephora) revealed many shared features. Taken together, these observations explain how CoA-dependent transferases with similar structural features can participate in different biochemical pathways across species. [ABSTRACT FROM AUTHOR]

Calsequestrin (CASQ) is a major Ca2+-storage/buffer protein present in the sarcoplasmic reticulum of both skeletal (CASQ1) and cardiac (CASQ2) muscles. CASQ has significant affinity for a number of pharmaceutical drugs with known muscular toxicities. Our approach, with in silico molecular docking, single crystal X-ray diffraction, and isothermal titration calorimetry (ITC), identified three distinct binding pockets on the surface of CASQ2, which overlap with 2-methyl-2,4-pentanediol (MPD) binding sites observed in the crystal structure. Those three receptor sites based on canine CASQ1 crystal structure gave a high correlation (R² = 0.80) to our ITC data. Daunomycin, doxorubicin, thioridazine, and trifluoperazine showed strong affinity to the S1 site, which is a central cavity formed between three domains of CASQ2. Some of the moderate-affinity drugs and some high-affinity drugs like amlodipine and verapamil displayed their binding into S2 sites, which are the thioredoxin-like fold present in each CASQ domain. Docking predictions combined with dissociation constants imply that presence of large aromatic cores and less flexible functional groups determines the strength of binding affinity to CASQ. In addition, the predicted binding pockets for both caffeine and epigallocatechin overlapped with the S1 and S2 sites, suggesting competitive inhibition by these natural compounds as a plausible explanation for their antagonistic effects on cardiotoxic side effects. [ABSTRACT FROM AUTHOR]

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. [ABSTRACT FROM AUTHOR]

Both cardiac and skeletal calsequestrin (CASQ2 and CASQ1) serve as a major Ca storage/buffer protein in the sarcoplasmic reticulum (SR) by sequestering and releasing large numbers of Ca ions during each muscular contraction and relaxation cycle. CASQ isolated from various species often exists in a phosphorylated form, but phosphorylation's role is not yet understood. Here, the authors identified two phosphorylation sites, Ser and Ser, for the first time, in human CASQ2 (hCASQ2) by mass-spectroscopy and evaluated the consequences of such phosphorylation. Substitution of these two serines with phosphoserine-mimicking aspartic-acid residues results in a significant increase in helical content, solubility and Ca-binding capacity above 6 mM [Ca]. However, neither substitution of Ser nor Ser alone produce any significant changes. Based on the crystal structures of hCASQ2, Ca binding capacity data, turbidity, and light scattering profiles, it was propose that phosphorylation at these two positions produces a disorder-to-order or coil-to-helix transition of the C-terminus, which in turn provides a more stable network of polyanions. Therefore, considering all the previous reports and the new data, the observed dynamic in vivo phosphorylation of CASQ could provide the basis not only for effective regulation of Ca buffering capacity, but also for the junctional SR trafficking mechanism. [ABSTRACT FROM AUTHOR]

This research adopts the utilitarian view of volunteering as a starting point: we posit that for an undergraduate student population volunteering is motivated by career enhancing and job prospects. We hypothesize that in those countries where volunteering signals positive characteristics of students and helps advance their careers, their volunteer participation will be higher. Furthermore, regardless of the signaling value of volunteering, those students who volunteer for utilitarian reasons will be more likely to volunteer but will exhibit less time-intensive volunteering. Using survey data from 12 countries (n = 9,482), we examine our hypotheses related to motivations to volunteer, volunteer participation, and country differences. Findings suggest that students motivated to volunteer for building their résumés do not volunteer more than students with other motives. However, in countries with a positive signaling value of volunteering, volunteering rates are significantly higher. As expected, students motivated by résumé building motivations have a lower intensity of volunteering. [ABSTRACT FROM AUTHOR]

Sphingobium chlorophenolicum completely mineralizes PCP (pentachlorophenol). Two GSTs (glutathione transferases), PcpC and PcpF, are involved in the degradation. PcpC uses GSH to reduce TeCH (tetrachloro-p-hydroquinone) to TriCH (trichloro-p-hydroquinone) and then to DiCH (dichloro-p-hydroquinone) during PCP degradation. However, oxidatively damaged PcpC produces GS-TriCH (S-glutathionyl-TriCH) and GS-DiCH (S-glutathionyl-TriCH) conjugates. PcpF converts the conjugates into TriCH and DiCH, re-entering the degradation pathway. PcpF was further characterized in the present study. It catalysed GSH-dependent reduction of GS-TriCH via a Ping Pong mechanism. First, PcpF reacted with GS-TriCH to release TriCH and formed disulfide bond between its Cys53 residue and the GS moiety. Then, a GSH came in to regenerate PcpF and release GS–SG. A TBLASTN search revealed that PcpF homologues were widely distributed in bacteria, halobacteria (archaea), fungi and plants, and they belonged to ECM4 (extracellular mutant 4) group COG0435 in the conserved domain database. Phylogenetic analysis grouped PcpF and homologues into a distinct group, separated from Omega class GSTs. The two groups shared conserved amino acid residues, for GSH binding, but had different residues for the binding of the second substrate. Several recombinant PcpF homologues and two human Omega class GSTs were produced in Escherichia coli and purified. They had zero or low activities for transferring GSH to standard substrates, but all had reasonable activities for GSH-dependent reduction of disulfide bond (thiol transfer), dehydroascorbate and dimethylarsinate. All the tested PcpF homologues reduced GS-TriCH, but the two Omega class GSTs did not. Thus PcpF homologues were tentatively named S-glutathionyl-(chloro)hydroquinone reductases for catalysing the GSH-dependent reduction of GS-TriCH. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/ PO4--dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, α-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers. [ABSTRACT FROM AUTHOR]

The crystal structure of (4S)-limonene synthase from Mentha spicata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-Å resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate. respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate-metal ion coordination and substrate binding conformation in the course of the multistep reaction. [ABSTRACT FROM AUTHOR]

Ionizing radiation and doxorubicin both produce oxidative damage and double-strand breaks in DNA. Doublestrand breaks and oxidative damage are highly toxic and cause cell cycle arrest, provoking DNA repair and apoptosis in cancer cell lines. To investigate the response of normal human cells to agents causing oxidative damage, we monitored alterations in gene expression in F65 normal human fibroblasts. Treatment with γ-irradiation and doxorubicin altered the expression of 23 and 68 known genes, respectively, with no genes in common. Both agents altered the expression of genes involved in cell cycle arrest, and arrested the treated cells in G2/M phase 12 h after treatment. 24 h after γ-irradiation, the percentage of G1 cells increased, whereas after doxorubicin treatment the percentage of G2/M cells remained constant for 24 h. Our results suggest that F65 cells respond differently to γ-irradiation- and doxorubicininduced DNA damage, probably using entirely different biochemical pathways. [ABSTRACT FROM AUTHOR]

Rubredoxin is a small iron-sulfur (FeS4) protein involved in oxidation–reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states. Despite the lack of the gating side chain in this protein, the structure of the reduced [L41A] rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin. In contrast, both the wild-type and [L41A] rubredoxins in the oxidized state do not have water molecules in this location. The reduction potential of the [L41A] variant was ~50 mV more positive than wild-type. Based on these observations, it is proposed that the site around the Sγ of Cys9 serves as a port for an electron acceptor. Lastly, the Fe–S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between Sγ of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart. This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent. [ABSTRACT FROM AUTHOR]

It is well known that exposure to UV induces DNA damage, which is the first step in mutagenesis and a major cause of skin cancer. Among a variety of photoproducts, cyclobutane-type pyrimidine photodimers (CPD) are the most abundant primary lesion. Despite its biological importance, the precise relationship between the structure and properties of DNA containing CPD has remained to be elucidated. Here, we report the free (unbound) crystal structure of duplex DNA containing a CPD lesion at a resolution of 2.0 Å. Our crystal structure shows that the overall helical axis bends ≈30° toward the major groove and unwinds ≈9°, in remarkable agreement with some previous theoretical and experimental studies. There are also significant differences in local structure compared with standard B-DNA, including pinching of the minor groove at the 3' side of the CPD lesion, a severe change of the base pair parameter in the 5 ' side, and serious widening of both minor and major groves both 3 ' and 5 'of the CPD. Overall, the structure of the damaged DNA differs from undamaged DNA to an extent that DNA repair proteins may recognize this conformation, and the various components of the replicational and transcriptional machinery may be interfered with due to the perturbed local and global structure. [ABSTRACT FROM AUTHOR]

Vacuolar sorting receptor (VSR) proteins bind soluble protein ligands in a sequence-specific manner and target them to the lytic vacuole in plant cells. A VSR from Arabidopsis thaliana, AtBP80b, has been successfully purified after heterologous expression in Drosophila S2 cells. The AtBP80b protein (560 amino acids) was crystallized by the hanging-drop method with a PEG 400-based precipitant. Preliminary X-ray diffraction studies of an AtBP80b crystal showed that it belongs to the cubic space group P213 (or P4232) and has unit-cell parameters a = b = c = 145.9 Å. Crystals of the VSR diffract beyond 2.5 Å resolution. [ABSTRACT FROM AUTHOR]

Examines the cloning of direct oxygen-sensor (DOS) protein in Escherichia coli. Characterization of the DOS proteins; Absorption properties of DOS heme; Composition of the oxygen-sensing protein subgroup.

Although bioinformatics, the use of computational science to study biology, has become imperative in many areas of the biological sciences and related career paths, introductory biochemistry courses may disregard practical knowledge on bioinformatics. For this reason, we merged a hands-on activity module into an undergraduate biochemistry course in two ways. First, we incorporated bioinformatics modules for building phylogenetic trees by aligning the active sites of 10 chosen related α-amylase enzymes using freely available data. Secondly, we chose three of those 10 α-amylase enzymes to compare the 3D structure of their active sites. This module gives the students an opportunity to understand how to access biological information from public databases such as GenBank, and analyze the information using software like MEGA, ClustalW, and RasMol. Overall, our module should provide instructors with ideas on how to develop similar modules and encourage students to develop further independence in the use of bioinformatics tools. [ABSTRACT FROM AUTHOR]

At a time when antibiotic resistance is seemingly ubiquitous worldwide, understanding the mechanisms responsible for successful emergence of new resistance genes may provide insights into the persistence and pathways of dissemination for antibiotic-resistant organisms in general. For example, Escherichia coli strains harboring a class A b-lactamase-encoding gene (blaCTX-M-15) appear to be displacing strains that harbor a class C b-lactamase gene (blaCMY-2) in Washington State dairy cattle. We cloned these genes with native promoters into low-copy-number plasmids that were then transformed into isogenic strains of E. coli, and growth curves were generated for two commonly administered antibiotics (ampicillin and ceftiofur). Both strains met the definition of resistance for ampicillin ($32 mg/mL) and ceftiofur ($16 mg/mL). Growth of the CMY-2-producing strain was compromised at 1,000 mg/mL ampicillin, whereas the CTX-M-15-producing strain was not inhibited in the presence of 3,000 mg/mL ampicillin or with most concentrations of ceftiofur, although there were mixed outcomes with ceftiofur metabolites. Consequently, in the absence of competing genes, E. coli harboring either gene would experience a selective advantage if exposed to these antibiotics. Successful emergence of CTX-M-15-producing strains where CMY-2-producing strains are already established, however, requires high concentrations of antibiotics that can only be found in the urine of treated animals (e.g., .2,000 mg/mL for ampicillin, based on literature). This ex vivo selection pressure may be important for the emergence of new and more efficient antibiotic resistance genes and likely for persistence of antibiotic-resistant bacteria in food animal populations. [ABSTRACT FROM AUTHOR]

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3'-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC30H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide-binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP+ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering. [ABSTRACT FROM AUTHOR]

The article focuses on a study which detects parallelism between echolocating bats and toothed whales in proteins that could contribute to high-frequency hearing adaptations. It mentions echolocating mammals exhibit an unusually high number of parallel substitutions in fast-twitch muscle fiber proteins. It also mentions parallel substitutions are expressed in the superfast sound-producing muscle of bats and calcium-sequestering polymers at lower calcium concentrations.

Calsequestrin 1 is the principal Ca2+ storage protein of the sarcoplasmic reticulum of skeletal muscle. Its inheritable D244G mutation causes a myopathy with vacuolar aggregates, whereas its M87T "variant" is weakly associated with malignant hyperthermia. We characterized the consequences of these mutations with studies of the human proteins in vitro. Equilibrium dialysis and turbidity measurements showed that D244G and, to a lesser extent, M87T partially lose Ca2+ binding exhibited by wild type calsequestrin 1 at high Ca2+ concentrations. D244G aggregates abruptly and abnormally, a property that fully explains the protein inclusions that characterize its phenotype. D244G crystallized in low Ca2+ concentrations lacks two Ca2+ ions normally present in wild type that weakens the hydrophobic core of Domain II. D244G crystallized in high Ca2+ concentrations regains its missing ions and Domain II order but shows a novel dimeric interaction. The M87T mutation causes a major shift of the α-helix bearing the mutated residue, significantly weakening the back-to-back interface essential for tetramerization. D244G exhibited the more severe structural and biophysical property changes, which matches the different pathophysiological impacts of these mutations. [ABSTRACT FROM AUTHOR]

Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-β-lactamase superfamily and play critical roles in animals, plants and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5 and 2.5 Å resolution, respectively. The structures of both PDOs were homo-dimers, and their metal centers and β-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-β-lactamase superfamily, but also provide detailed information on substrate binding by PDOs. [ABSTRACT FROM AUTHOR]

Glutathionyl-hydroquinone reductases (GS- HQRs) are a newly identified group of glutathione transferases, and they are widely distributed in bacteria, halobacteria, fungi, and plants. GS-HQRs catalyze glutathione (GSH)-dependent reduction of glutathionyl-hydroquinones (GS-hydroquinones) to hydroquinones. GS-hydroquinones can be spontaneously formed from benzoquinones reacting with reduced GSH via Michael addition, and GS-HQRs convert the conjugates to hydroquinones. In this report we have determined the structures of two bacterial GS-HQRs, PcpF of Sphingobium chlorophenolicum and YqjG of Escherichia coli. The two structures and the previously reported structure of a fungal GS-HQR shared many features and displayed complete conservation for all the critical residues. Furthermore, we obtained the binary complex structures with GSmenadione, which in its reduced form, GS-menadiol, is a substrate. The structure revealed a large H-site that could accommodate various substituted hydroquinones and a hydrogen network of three Tyr residues that could provide the proton for reductive deglutathionylation. Mutation of the Tyr residues and the position of two GSH molecules confirmed the proposed mechanism of GS-HQRs. The conservation of GS-HQRs across bacteria, halobacteria, fungi, and plants potentiates the physiological role of these enzymes in quinone metabolism. [ABSTRACT FROM AUTHOR]

Calsequestrin (CEQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca2+-dependent manner. However, no direct evidence of CEQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CEQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca2+ that were decondensed upon Ca2+ depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca2+ depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca2+ concentration, implying that JNT is an essential component of the CEQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca2+ and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CEQ are critical in SR Ca2+ homeostasis. [ABSTRACT FROM AUTHOR]

The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants. [ABSTRACT FROM AUTHOR]

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN·FMN complex, and FMN·NADH complex of EmoB were determined at 2.5Å resolution. The structure of EmoB is a homotetramer consisting of four α/β-single-domain monomers of five parallel β-strands flanked by five α-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN·FMN and FMN·NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN. [ABSTRACT FROM AUTHOR]

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabiclopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADTI (Atlgl1790), ADT2 (At3g07630), and ADT6 (Atlg08250), more efficiently utilized arogeuate than prephenate, whereas the remaining three, ADT3 (At2927820), ADT4 (At3944720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had kcat/Km values of 1050, 7650, and 1560 M-1 S-1 for arogenate versus 38, 240, and 16 M-1 s-1 for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had kcat/Km values of 1140, 490, and 620 M-1 s-1, with prephenate not serving as a substrate unless excess recombinant protein (>150 μg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6. [ABSTRACT FROM AUTHOR]