Unusual Oligomeric Laccase-like Oxidases from Ascomycete Curvularia geniculata VKM F-3561 Polymerizing Phenylpropanoids and Phenolic Compounds under Neutral Environmental Conditions

Abstract

The unique oligomeric alkaliphilic laccase-like oxidases of the ascomycete C. geniculata VKM F-3561 (with molecular masses about 1035 and 870 kDa) were purified and characterized for the first time. The ability of the enzymes to oxidize phenylpropanoids and phenolic compounds under neutral environmental conditions with the formation of previously unknown di-, tri-, and tetrameric products of transformation was shown. The possibility to obtain industrially valuable compounds (dihydroxybenzyl alcohol and hydroxytyrosol) from caffeic acid using laccase-like oxidases of C. geniculata VKM F-3561 has been shown. Complete nucleotide sequence of the laccase gene, which is expressed at the peak of alkaliphilic laccase activity of the fungus, and its promoter region were determined. Based on the phylogenetic analysis of the nucleotide sequence, the nearest relationship of the isolated laccase gene with similar genes of fungi of the genera Alternaria, Bipolaris, and Cochliobolus was shown. Homologous model of the laccase structure was predicted and a proton channel was found, which was presumably responsible for the accumulation and transport of protons to T2/T3-copper center in the alkaliphilic laccase molecule and providing the functional activity of the enzyme in the neutral alkaline environment conditions.

Keywords: alkaliphilic laccase; ascomycete; laccase-like oxidase; neutral environmental conditions; phenolic compounds; phenylpropanoids; polymerization; proton channel; transformation.

Figure 8

Superposition of the T1 centers of the laccases from M. roridum VKM F-3565 (top caption, salmon texture), C. geniculata VKM F-3561 (middle caption, violet purple texture), laccase-like bacterial protein CotA from Bacillus subtilis (RCSB PDB acc. no.: 1GSK, lower signature, gray structure, (A) or laccase from the fungus Trametes versicolor (RCSB PDB acc. no.: 1KYA, lower signature, gray structure, (B). The position of the axial methionine residue located near the T1-copper coordination sphere of bacterial laccases [84] is highlighted in blue. The position of the aspartate residue involved in the deprotonation of phenolic substrates in the T1-center cavity of the laccase of Trametes versicolor [86] is marked by the red frame.

Figure 1

Dynamics of laccase activity of the fungus C. geniculata VKM F-3561 during submerged cultivation in the presence of various concentrations of the mixture of ground seeds of 5 cereals as sole sources of carbon and energy (A), as well as in the presence of 100 g/L mixture of the ground seeds and various concentrations of copper sulfate (B).

Figure 2

Determination of molecular ((A), by gel filtration on Superdex 200) and subunit ((B), by SDS-electrophoresis (7% PAGE)) masses of the purified fungal laccase-like oxidases of C. geniculata VKM F-3561. Here, 1035 kDa and 870 kDa are approximate molecular masses of the oxidase I and the oxidase II, respectively. The masses of the standards (45 kDa—ovalbumin, 66 kDa—bovine serum albumin, 150 kDa—alcohol dehydrogenase, 443 kDa—apoferritin, 669 kDa—thyreoglobulin, and 2000 kDa—dextran blue) are shown in black circles. 1—The glycosylated oxidase I, 2—the glycosylated oxidase II, M—standards, 3—the deglycosylated form of the oxidase I, 4—the deglycosylated form of the oxidase II.

Figure 3

UV-vis absorption spectra of the purified glycosylated oxidases of C. geniculata VKM F-3561 in 20 mM Na-acetate buffer at pH 5.0.

Figure 4

Phylogenetic relationships between the laccase gene of C. geniculata VKM F-3561 and the genes of known laccases inferred under the Maximum Likelihood criterion. Bootstrap values are placed near the corresponding nodes. Ascomycetes are marked in light red, basidiomycetes are in light blue. The sequence of the laccase gene of C. geniculata VKM F-3561 is marked with a red circle.

Figure 5

Phylogenetic relationships between the laccase from C. geniculata VKM F-3561 and all laccases with known structure based on the alignment of their amino acid sequences (RCSB PDB acc. numbers of proteins are indicated). The bootstrap values (consensus support, %) are displayed on the tree. The red filled circle marks the sequence of the laccase of C. geniculata VKM F-3561, the orange filled circles mark sequences of laccases which are more active with phenolic compounds at pH ≥ 7.0. The orange empty circles indicate the laccases, which are more active at pH values around 6.0. The laccases with maximum activity at acidic conditions (pH ≤ 5.0) are marked by the black empty circles, and the laccases with unknown pH optimum are marked by the grey filled circles. The laccases from ascomycetous fungi are marked in light red shadow, the basidiomycetous laccases are marked in light blue shadow, and the bacterial laccases are marked in light orange shadow.

Figure 6

Regulatory elements in the promoter region of the laccase gene of the fungus C. geniculata VKM F-3561 isolated from mRNA at the peak of the fungal alkaliphilic laccase activity. HSE—heat shock responsive element, XRE—xenobiotic responsive element, ACE—element activating gene transcription in response to monovalent metals induction), STRE—stress response element, CreA—cAMP mediated glucose repression, NIT2—nitrogen repression response element, pH dependent element PacC, ATATC-element.

Figure 7

The structure of the laccase of C. geniculata VKM F-3561 calculated by homology modeling: (A)—the overall structure of the laccase molecule, (B)—the laccase copper sites. Copper atoms are marked in cyan.

Figure 9

The alignment of amino acid sequences of the alkaliphilic laccases from C. geniculata VKM F-3561 (F-3561_20d) and M. roridum VKM F-3565 (F-3565_lac) containing signal peptides ((A), marked in orange) and T2-, T1/T3α-, and proton channels in the structure of C. geniculata VKM F-3561 laccase (B). The copper atoms are marked in blue. The aspartate residues presumably involved in proton transfer during a catalytic process are colored in light green.

Figure 10

Connolly accessible surface representations colored according to electrostatic potential (−5 keV, red; +5 keV, blue) for laccase of C. geniculata VKM F-3561 calculated at pH 5.0 (A,C) and pH7.0 (B,D). (A,B) are external views, (C,D) are internal sections of the molecule. Copper atoms are highlighted in light brown.