LccA, an archaeal laccase secreted as a highly stable glycoprotein into the extracellular medium by Haloferax volcanii

Abstract

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 +/- 10 mU.ml(-)(1)) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U.mg(-)(1) with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO(4). Optimal oxidation of ABTS and SGZ was at 45 degrees C and pH 6 and pH 8.4, respectively. The apparent K(m) values for SGZ, bilirubin, and ABTS were 35, 236, and 670 muM, with corresponding k(cat) values of 22, 29, and 10 s(-)(1), respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50 degrees C of 31.5 h.

FIG. 1.

Sequence alignment of H. volcanii LccA (Hvo_B0205) to multicopper oxidases. The vertical lines represent cleavage sites with experimental evidence based on the N-terminal sequence of the mature LccA (HVO_B0205) and Myrothecium verrucaria bilirubrin oxidase (Mver_Q12737) (37). TAT motifs, indicated by a double underline, are based on in silico analysis using the TatP 1.0 server. Note that TatP also predicts both cleavage sites described above. Residues that bind the four copper atoms to form the type 1, 2, and 3 copper sites of known multicopper oxidases are indicated by dots above the amino acid residues. Potential O- and N-linked glycosylation sites with EnsembleGly scores above 0.8 are indicated by O* and N*, respectively, above the residues, with the Thr-X₅-Pro-X-Pro and Asn-X-Thr motifs (where X represents any amino acid) boxed. Amino acid sequences in boldface and underlined represent peptides detected by MS-MS analysis of the laccase purified from H. volcanii US02 (details of MS-MS analysis are shown in the supplemental material). HVO_B0205, H. volcanii laccase LccA (this study); Hlac_ZP-02016827, Halorubrum lacusprofundi putative multicopper oxidase (GenBank accession no. 153896174); Bsub_BAA22774, Bacillus subtilis spore coat protein A (CotA) with laccase activity (28, 43); Scha_AAY23005, Stachybotrys chartarum phenol oxidase A (42); Mver_Q12737, M. verrucaria bilirubrin oxidase (37).

FIG. 2.

Laccase activity of H. volcanii SB01 and US02 strains at various stages of growth. H. volcanii SB01 (A), US02 (B), and the parent, H26 (data not shown), were inoculated to a final optical density at 600 nm (OD₆₀₀) of 0.01 in medium supplemented with novobiocin and 100 μM copper sulfate. LMM and YPC were optimal for production of laccase activity for SB01 and US02, respectively. The cells were grown for up to 120 h at 42°C in an orbital shaker (150 to 200 rpm). Samples were withdrawn at intervals and used for estimation of growth (1 OD₆₀₀ unit ≈ 1 × 10⁹ CFU per ml) and assay of laccase activity. For analysis of cell lysates, the cells were harvested by centrifugation (10 min at 10,000 × g and 4°C), washed twice in T buffer supplemented with 2 M NaCl, resuspended in T buffer, and lysed by sonication (15 s on, 45 s off at 20% amplitude for 3 min). The culture broth (filtered at 0.4 μm) and cell lysate were assayed for laccase (SGZ-oxidizing) activity under standard conditions. Data are presented as means ± standard deviations.

FIG. 3.

LccA purified to electrophoretic homogeneity from the culture broth of H. volcanii US02. Shown is reducing 10% SDS-PAGE of LccA at various stages of purification, including ethanol precipitate (EP) (lane 1) and MonoQ 10/10, pH 8.4 (MQ) (lane 2), as indicated. Molecular mass standards are on the left. Proteins were stained with Coomassie brilliant blue R-250.

FIG. 4.

LccA purified from US02 is a glycoprotein. (A) Native gels of LccA at its final stages of purification from US02 (Superdex 200 HR 10/30, lanes 1 and 2) and SB01 (MonoQ 10/10, pH 8.4, lanes 3 and 4). The gels were stained for total protein with Coomassie brilliant blue R-250 (CB) and for laccase activity by in-gel oxidation of SGZ (SGZ), as indicated. Prestained kaleidoscope and low-range protein standards (Bio-Rad) were included for US02 and SB01, respectively, as indicated at the left of each gel. (B) Pro-Q Emerald glycoprotein analysis of US02-purified LccA. Proteins were separated by reducing 10% SDS-PAGE. The gels were stained for glycosylation with Pro-Q Emerald (left) and for total protein with Sypro-Ruby (right), as indicated. The proteins included carbonic anhydrase (CA) (lanes 1); T. versicolor laccase (TvLc) (lanes 2); LccA MonoQ 10/10, pH 8.4, fractions (HvLc_MQ) (lanes 3); LccA ethanol precipitate (HvLc_EP) (lanes 4); and CandyCane molecular mass standards (Molecular Probes), including a mixture of glyosylated (G) and nonglycosylated proteins, as indicated (lanes 5). (C) Deglycosylation of LccA. LccA purified from US02 was treated with TFMS on ice for 0 h (lanes 1), 10 h (lanes 2), and 3 h (lane 3) and separated by reducing 6 and 10% SDS-PAGE, as indicated (see Materials and Methods for details).

FIG. 5.

UV-visible absorbance spectrum of LccA. The absorbance of LccA (1.5 mg·ml⁻¹) purified from H. volcanii US02 in T buffer containing 185 mM NaCl was analyzed from 300 to 700 nm.