Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2.

Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 +/- 0.004 microM(-1) x s(-1) for ATP A2, respectively. The structural origin of the differential reactivity is discussed in relation to glycosylation and amino acid substitutions. The results are of general importance to the use of homologous models and structure determination at low temperatures.

Peroxidase production in vitro by Armoracia lapathifolia (horseradish)-transformed root cultures: effect of elicitation on level and profile of isoenzymes.

Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402. They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field-grown species of Armoracia sp. The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase. At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors. The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx. 100% in peroxidase activity. The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures. Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.

Identification of a critical phenylalanine residue in horseradish peroxidase, Phe179, by site-directed mutagenesis and 1H-NMR: implications for complex formation with aromatic donor molecules.

The functional and structural significance of Phe179 of horseradish peroxidase isoenzyme C (HRP C) has been investigated by site-directed mutagenesis. This residue is located in a structurally variable insertion between helices F and G, a motif unique to peroxidases of higher plants. Results obtained for three recombinant enzymes, with Phe179 substituted by Ala, His, or Ser, provide the first demonstration of the importance of this side chain for the binding of aromatic donor molecules. Experimental parameters for direct comparison with the wild-type enzyme were obtained by extensive solution state characterization using both optical and 1H-NMR spectroscopy. Significant chemical shift variations for resonances associated with the exposed heme edge, notably heme methyl C18H3 and heme propionate C17(1)H2, were recorded in NMR spectra of both the resting and cyanide-ligated states of the three Phe179 mutants. Furthermore, comparison of NOE connectivities in NOESY spectra of cyanide-ligated wild-type and mutant enzymes enabled the elusive assignment of the aromatic side chain in close proximity to heme methyl C18H3 to be made to Phe179. Replacement of Phe179 by Ala resulted in an 80-fold decrease in the binding affinity of the cyanide-ligated enzyme for benzhydroxamic acid, with a Kd value similar to that determined for cyanide-ligated HRP A2 (an acidic isoenzyme with valine at position 179). The binding affinity of Phe179-->Ser was similarly decreased, while that of Phe179-->His was partially restored relative to wild-type HRP C. Cyanide-ligated Phe179-->His HRP C exhibited a unique pH-dependent spectral transition associated with a pKa value of 6.5 +/- 0.2, assigned to the His179 side chain. Two closely related enzyme forms exhibiting different affinities for benzhydroxamic acid were observed at neutral pH and above, indicating that the protonation state of His179 gave rise to microheterogeneity in the aromatic donor molecule binding site.