Molecular analysis of a perchlorate reductase from a perchlorate-respiring bacterium Perc1ace

Author： Okeke Benedict C. Frankenberger Jr. William T.

ISSN： 0944-5013

Source： Microbiological Research, Vol.158, Iss.4, 2003-12, pp. : 337-344

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Abstract

Perchlorate (ClO₄^–) is a major ground water pollutant of public health concern. ClO₄^– reductase is the key enzyme in the pathway of ClO₄^– breakdown. ClO₄^– reductase from cell-free extracts of the ClO₄^–-respiring bacterium perc1ace was purified 10-fold by ion-exchange and molecular exclusion fast protein liquid chromatography (FPLC). The ClO₄^– reductase catalyzed the reduction of ClO₄^– at a V_max and K_m of 4.8 U mg protein^–1 and 34.5 M, respectively. ClO₄^– reduction was achieved in the temperature range of 20 to 40C and with optimum activity at 25C to 30C and pH 7.5 to 8.0. Molecular masses of two subunits of ClO₄^– reductase were determined by SDS-PAGE to be 35 kDa and 75 kDa. MALDI-TOF/MS analysis of a trypsin digest of the 35 kDa subunit, revealed several tryptic peptides. Amino acid sequences of 22 tryptic peptides of the 35 kDa ClO₄^– reductase subunit were obtained by electrospray mass spectrometry. GenBank protein Blast analysis of the amino acid sequences revealed relevant similarity to reductases, dehydrogenases and heme proteins. Data obtained are useful towards the identification of the overall genetic determinants of ClO₄^– reduction and specific in situ detection of ClO₄^– as well as NO₃-reducing bacteria in ground water.