HOW DEEP IS YOUR BLUE? A NOVEL COPPER CONTAINING NITRITE REDUCTASE FROM A THERMOPHILIC BACTERIUM ISOLATED FROM THE SOUTH AFRICAN DEEP SURFACE

FERRONI FM; FLORENCIA BELÉN SCHMIDT; BRONDINO CD; OPPERMAN, D.J.

Buenos Aires

Congreso; JOINT MEETING OF BIOSCIENCE SOCIETIES. LIII ANNUAL MEETING OF ARGENTINE SOCIETY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (SAIB); 2017

many microorganisms of the genus Thermus have been isolated from hot environments around the world but particularly Thermus scotoductus SA-01 was isolated from fissure water collected 3.2 km below the surface in a South African gold mine. Denitrification is a relevant energy pathway in this kind of microorganisms and the presence of both a cytochrome cd1 (NirS) and a copper containing nitrite reductase (NirK) is exceptional. The genome of T. scotoductus SA-01 showed the presence of genes for both enzymes. Here we present the expression, purification and the structural and functional characterization of NirK from T. scotoductus SA-01 (TsNirK). Escherichia coli BL21(DE3) Gold cells were used to express the optimized Tsc_c17620 gene inserted into pET22b(+). The recombinant TsNirK was purified by several chromatographic steps and the turquoise pure enzyme showed different spectroscopic features from those observed in green/blue NirKs. Gel filtration analysis showed an elution peak corresponding to a homotrimer and metal analysis reported 3 mol copper per mol monomer. We proceeded on protein crystallization screening assays and TsNirK crystals developed in several conditions. Single crystals grown in 0.2 M CaCl2.2 H2O 0.1 M HEPES sodium pH: 7.5 28% w/v PEG 400 and diffracted at 1.63 Å (beamline I04-I, Diamond Synchrotron, UK). The obtained overall structure reveals a homotrimer with 3 copper atoms per each subunit. The catalytic type 2 copper center (T2Cu) is 12.6 Å His-Cys bridged to a non-common type 1 copper center (T1CuN-ter). An extra cupredoxin domain is located at the C termini of each monomer and binds a normal type 1 copper center (T1CuC-ter) that resembles amicyanin. Thermal stability, kinetic properties and structural features make this enzyme a good candidate for the development of nitrite biosensors.