Jörg Nüske

Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes

ABSTRACT Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H2O2 (2 mM) for optimum activity. The apparent Km values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H2O2 were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.

Large scale production of manganese-peroxidase using agaric white-rot fungi

The agaric basidiomycetes Nematoloma (Hypholoma) frowardii strain b19 and Clitocybula dusenii strain b11 were used to produce manganese peroxidase (MnP) in 5-, 30- and 300-liter stirred-tank bioreactors. Large amounts of MnP (up to 2000 U l−1) were gained by using sodium acetate as the major carbon source and by supplementing Mn2+ to stimulate the enzyme production. In media containing both acetate and glucose, fungi preferred always acetate to glucose as actual carbon source. Both fungi secreted oxalic acid (up to 8 mM) into the medium during the fermentation process. There was no loss in productivity and no significant changes in the isoenzyme pattern during scale up from 5 to 300-liter fermentors. A cyclic batch process has been developed keeping the fungal cultures in the productive state for prolonged time and allowing to harvest the enzyme containing culture filtrate every two to three days.

3‐Methylarginine from Pseudomonas syringae pv. syringae 22d/93 Suppresses the Bacterial Blight Caused by Its Close Relative Pseudomonas syringae pv. glycinea

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L‐arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay‐guided fractionation using ion‐exchange chromatography and subsequent RP‐HPLC fractionation. The structure of the natural product was identified by HR‐ESI‐MS, HR‐ESI‐MS/MS, and NMR spectroscopy experiments as 3‐methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3‐methylarginine is the introduction of the methyl group by a S‐adenosylmethionine (SAM)‐dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM‐dependent methyltransferase of the 3‐methylarginine biosynthesis to be identified.

Fluoride-Dependent Conversion of Organic Compounds Mediated by Manganese Peroxidases in the Absence of Mn2+ Ions

Manganese peroxidase generally mediates the oxidation of Mn(2+) to Mn(3+) with H(2)O(2) as an oxidant. Several manganese peroxidases purified from different lignin-degrading fungi were found to mediate a fluoride-dependent conversion of organic substrates such as monochlorodimedone or 2,6-dimethoxyphenol in the absence of manganese ions. Using the manganese peroxidase MnP-1 from Bjerkandera adusta strain Ud1, these fluoride-dependent reactions were studied with respect to different substrates converted, reaction products, and kinetic properties to shed some light on the reaction mechanism of manganese peroxidase. The analysis of the reaction products formed from monochlorodimedone and 2,6-dimethoxyphenol showed that the substrates were oxidized rather than fluorinated. The addition of fluoride to MnP-1 resulted in altered absorption spectra, indicating a coordinative binding of fluoride or HF to the heme iron; the fluoride:heme stoichiometry was determined to be 1:1 and the K(D) value to be approximately 2.5 mM at pH 3.4. The high K(D) value indicates weak binding of fluoride to the heme. Fluoride appeared to act as a partially competitive inhibitor with respect to hydrogen peroxide for binding to the heme as the sixth ligand. From the findings, a putative model for the fluoride-dependent reaction was developed. The data were interpreted to indicate that changes of the reaction center of manganese peroxidase as, for example, caused by fluoride binding may lead to the oxidation of organic compounds in the absence of manganese by opening a long-range electron transfer pathway.