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 ⅐ 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 ⅐ 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°C and pH 6 and pH 8.4, respectively. The apparent K m values for SGZ, bilirubin, and ABTS were 35, 236, and 670 M, 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°C of 31.5 h.Multicopper oxidases (MCOs) are a family of enzymes that include laccases (p-diphenol: dioxygen oxidoreductases; EC 1.10.3.2), ascorbate oxidases (EC 1.10.3.3), ferroxidases (EC 1.16.3.1), bilirubin oxidases (EC 1.3.3.5), and other enzyme subfamilies (27,65). MCOs couple the oxidation of organic and/or inorganic substrates to the four-electron reduction of molecular oxygen to water. These enzymes often have four Cu atoms classified into type 1 (T1), type 2 (T2), and type 3 (T3) centers, in which a mononuclear T1 center on the surface of the enzyme provides long-range intramolecular one-electron transfer from electron-donating substrates to an internal trinuclear T2-T3 center formed by a T2 Cu coordinated with a T3 Cu pair. The T2-T3 cluster subsequently reduces dioxygen to water.Enzymes of the laccase subfamily oxidize a broad range of compounds, including phenols, polyphenols, aromatic amines, and nonphenolic substrates, by one-electron transfer to molecular oxygen and thus have a wide variety of applications from biofuels to human health. The best-known application is the use of a laccase from the lacquer tree Rhus vernicifera in paint and adhesives for more than 6,000 years in East Asia (29). Laccases have also been used in the delignification of pulp, bleaching of textiles and carcinogenic dyes, detoxification of water and soils, removal of phenolics from wines, improving adhesive properties of lignocellulosic products, determination of bilirubin levels in serum, and transformation of antibiotics and steroids (60). In addition, laccases have demonstrated pote...

Activity and Transcriptional Regulation of Bacterial Protein-Like Glycerol-3-Phosphate Dehydrogenase of the Haloarchaea in Haloferax volcanii

“…However, P1 glpA1 may serve as a nice complement to P tnaA for experiments requiring differential gene regulation. When glycerol was included in the medium, expression from P1 glpA1 was comparable to that from H. salinarum P2 rrnA , used routinely for protein production in recombinant strains of H. volcanii (18,30) (Table 2).…”

Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes

“…The apparent molecular masses of the two bands, 250 kDa and 180 kDa (28), are not far off from the predicted molecular masses for P. putida GB-1 MnxG (209 kDa) and McoA (123 kDa). Indeed, the size discrepancy may be due to potential N-glycosylation of MnxG and McoA, as has been reported for other MCOs in archaea and Saccharomyces cerevisiae (41,42), or due to incomplete denaturation, causing the proteins to run anomalously. (ii) The failure of all the previous genetic experiments to identify the Mn(II) oxidase (21)(22)(23)(24)(25) also suggested that there is more than one oxidase gene in P. putida GB-1, since it would be extremely difficult to simultaneously disrupt multiple oxidase genes through random mutagenesis.…”

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

“…HvPPA tolerance to temperature and organic solvents. Haloarchaeal proteins are notable for their stability at 40 to 65°C and for their high tolerance of organic solvents (58)(59)(60)(61)(62). Here we found HvPPA to be moderately thermostable, with a half-life of thermal inactivation of 2 h at 65°C, and to retain 82% activity after incubation for 2 h at 42°C (Fig.…”