A new group of aromatic prenyltransferases in fungi, catalyzing a 2,7-dihydroxynaphthalene 3-dimethylallyl-transferase reaction - PubMed (original) (raw)

A new group of aromatic prenyltransferases in fungi, catalyzing a 2,7-dihydroxynaphthalene 3-dimethylallyl-transferase reaction

Elisa Haug-Schifferdecker et al. J Biol Chem. 2010.

Abstract

Five fungal genomes from the Ascomycota (sac fungi) were found to contain a gene with sequence similarity to a recently discovered small group of bacterial prenyltransferases that catalyze the C-prenylation of aromatic substrates in secondary metabolism. The genes from Aspergillus terreus NIH2624, Botryotinia fuckeliana B05.10 and Sclerotinia sclerotiorum 1980 were expressed in Escherichia coli, and the resulting His(8)-tagged proteins were purified and investigated biochemically. Their substrate specificity was found to be different from that of any other prenyltransferase investigated previously. Using 2,7-dihydroxynaphthalene (2,7-DHN) and dimethylallyl diphosphate as substrates, they catalyzed a regiospecific Friedel-Crafts alkylation of 2,7-DHN at position 3. Using the enzyme of A. terreus, the K(m) values for 2,7-DHN and dimethylallyl diphosphate were determined as 324 +/- 25 microM and 325 +/- 35 microM, respectively, and k(cat) as 0.026 +/- 0.001 s(-1). A significantly lower level of prenylation activity was found using dihydrophenazine-1-carboxylic acid as aromatic substrate, and only traces of products were detected with aspulvinone E, flaviolin, or 4-hydroxybenzoic acid. No product was formed with l-tryptophan, l-tyrosine, or 4-hydroxyphenylpyruvate. The genes for these fungal prenyltransferases are not located within recognizable secondary metabolic gene clusters. Their physiological function is yet unknown.

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Figures

FIGURE 1.

FIGURE 1.

Reactions catalyzed by the bacterial enzymes CloQ, NphB, and SCO7190 and by the fungal aspulvinone dimethylallyltransferase. The genuine aromatic substrate of NphB in the biosynthesis of naphterpin is unknown; 2,7-dihydroxynaphthalene is one of the artificial substrates accepted in vitro. GPP, geranyl diphosphate.

FIGURE 2.

FIGURE 2.

A, sequence comparison of the bacterial prenyltransferases NphB, CloQ, and NovQ with the fungal proteins PtfAt, PtfBf, PtfSs, PtfPm, and PtfMc. For PtfAt, both the current sequence in the database (PtfAt-Or) and the corrected sequence (PtfAt) are displayed. The amino acids of NphB involved in the binding of the pyrophosphate moiety (▴, Lys119, Asn173, Tyr216, Arg228, and Lys284; over H2O ■, Tyr282) and in the coordination of the Mg2+ ion (●, Asp62) are marked. Likewise, the position of the lysine residue of CloQ and NovQ, suggested to functionally replace the positive charge of the Mg2+ ion, is marked (★). The sequence was created with Clustal W2 (22) and visualized by ESPript 2.2 (23). The sequence shows the secondary structure elements of NphB: α, α helices; η, 310 helices; β, β strands; TT, strict β turns. Strict sequence identity is shown by a black box with a white character, and similarity is shown by bold characters in a black frame. B, sequence identities (%) of the predicted gene products of the fungal genes _ptf_At, _ptf_Bf, _ptf_Ss, _ptf_Pm-S, and _ptf_Mc with the bacterial ABBA prenyltransferases NphB, CloQ, and NovQ. For _ptf_Pm, an N-terminally truncated version (_ptf_Pm-S) was used for this calculation (see text).

FIGURE 3.

FIGURE 3.

Purification of the fungal prenyltransferases PtfAt (A), PtfBf (B), and PtfSs (C) after expression in the form of His8-tagged fusion proteins. Lane 1, molecular mass standards; lane 2, total protein before IPTG induction; lane 3, total protein after IPTG induction; lane 4, soluble protein after IPTG induction; lane 5, insoluble protein after IPTG induction; lane 6, protein after Ni2+ affinity chromatography; lane 7, molecular mass standards; lane 8, protein after ion exchange chromatography. The calculated masses are 36.6 kDa for PtfAt, 36.7 kDa for PtfSs, and 36.5 kDa for PtfBf. The 12% polyacrylamide gel was stained with Coomassie Brilliant Blue R-250.

FIGURE 4.

FIGURE 4.

A, the aromatic prenylation reaction catalyzed by PtfAt. B, HPLC analysis of prenyltransferase assays with PtfAt. Detection: UV, 285 nm.

FIGURE 5.

FIGURE 5.

Determination of Km values of PtfAt for DMAPP and 2,7-dihydroxynaphthalene. In A, 2,7-diydroxynaphthalene was kept constant at 2 m

m

. In B, DMAPP was kept constant at 2 m

m

. Km and _k_cat values were determined by nonlinear regression, using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA).

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