Arginase from Bacillus caldovelox

Abstract

Nine Thermus strains, six thermophilic Bacillus species, six extremely thermophilic archaebacteria and other thermophilic bacteria were cultured in arginine-supplemented media and screened for arginase, arginine deiminase and arginine decarboxylase activity by a reversed-phase liquid chromatography method developed to detect these arginine-catabolizing enzymes in cell-free extracts. Thermus, Bacillus and Sulfolobus strains produced arginase, but arginine deiminase and arginine decarboxylase activities were only detected in two archaebacterial anaerobes and Clostridium thermohydrosulfuricum. Other enzymes of the arginase and arginine deiminase pathways were also present in these bacteria. Inducible arginases (EC 3.5.3.1) were purified from Bacillus caldovelox (DSM 411) and Thermus 4-1A (TRUCC 52) by (NH₄)₂SO₄ and protamine sulphate precipitations, DEAE Sepharose CL-6B ion exchange chromatography, Superose 12 GP-FPLC, Mono Q IE-FPLC and phenyl-Superose HI-FPLC. B. caldovelox arginase has a Mᵣ of ~176000 and a subunit Mᵣ of 32000, suggesting a hexameric subunit structure. It has an isoelectric point of 5.4, a pH optimum for activity of 9 (at 60°C in 0.1M CAPS/NaOH buffer) and requires a divalent cation for activity. The purified enzyme contains 1.2 Mn²⁺ ions and 0.17 Fe²⁺ ions per subunit and is activated ~30% by Mn²⁺. The enzyme has a Kₘ for arginine of 3.4mM at pH9 and 25mM at pH7 and hydrolyses L-canavanine at 4.7% and D-arginine and L-homoarginine at <1% of the rate seen for L-arginine. It is competitively inhibited by L-ornithine with a Kᵢ of 0.55mM at pH9 and 4.4mM at pH7 and has an E, of 46.3kJ/mole at pH9. The B. caldovelox arginase is thermostable with a half life of 105min at 95°C at pH7 in the presence of 0.5mM Mn²⁺ and 25mM aspartic acid. Mn²⁺ and chelating agents or proteins have a synergistic stabilizing effect and it is suggested that an excessive [Mn²⁺] causes inactivation in the absence of a chelating agent. The apoenzyme, prepared by chelation of metal ions at pH7 at 60°C or by incubation at pH4.4 at 20°C, retains its native oligomeric structure but is dissociated into subunits at pH2.5 at 20°C. The subunits reassociate at pH9.5 at 20°C in the absence of Mn²⁺ to form the native oligomer. The Thermus 4-1A arginase has a Mᵣ of ~167000 and a subunit Mᵣ of 33000. It has an isoelectric point of 5.1, a pH optimum of 9.3-9.8 and is activated by V0²⁺, Mn²⁺, Ni²⁺ and Cd²⁺ and less strongly by Co²⁺, Fe²⁺, Mo(III) and Mg²⁺. The purified enzyme contains 0.33 Mn²⁺ ions and 0.1 Fe²⁺ ions per subunit and has an Eₐ of 30.2kJ/mole at pH9.