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In planta, H2O2 is produced as a by-product of enzymatic reactions and during defense responses. Ascorbate peroxidase (APX) is a key enzyme involved in scavenging cytotoxic H2O2. Here, we report the crystal structure of cytosolic APX from sorghum (Sorghum bicolor) (Sobic.001G410200). While the overall structure of SbAPX was similar to that of other APXs, SbAPX uniquely displayed four bound ascorbates rather than one. In addition to the ɣ-heme pocket identified in other APXs, ascorbates were bound at the δ-meso and two solvent-exposed pockets. Consistent with the presence of multiple binding sites, our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Bound ascorbate at two surface sites established an intricate proton network with ascorbate at the ɣ-heme edge and δ-meso sites. Based on crystal structures, steady-state kinetics, and site-directed mutagenesis results, both ascorbate molecules at the ɣ-heme edge and the one at the surface are expected to participate in the oxidation reaction. We provide evidence that the H2O2-dependent oxidation of ascorbate by APX produces a C2-hydrated bicyclic hemiketal form of dehydroascorbic acid at the ɣ-heme edge, indicating two successive electron transfers from a single-bound ascorbate. In addition, the δ-meso site was shared with several organic compounds, including p-coumaric acid and other phenylpropanoids, for the potential radicalization reaction. Site-directed mutagenesis of the critical residue at the ɣ-heme edge (R172A) only partially reduced polymerization activity. Thus, APX removes stress-generated H2O2 with ascorbates, and also uses this same H2O2 to potentially fortify cell walls via oxidative polymerization of phenylpropanoids in response to stress.

 

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.

 

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.