Molecular analysis of a perchlorate reductase from a perchlorate-respiring bacterium Perc1ace.
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 perc lace 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 Vmax and Km of 4.8 U mg protein(-1) and 34.5 microM, respectively. ClO4- reduction was achieved in the temperature range of 20 to 40 degrees C and with optimum activity at 25 degrees C to 30 degrees C 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.
Okeke
Benedict C
BC
Department of Environmental Sciences, University of California, Riverside, CA 92521, USA.
Frankenberger
William T
WT
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001673:Biodegradation, Environmental; D005618:Fresh Water / Q000382:microbiology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010472:Perchlorates / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014873:Water Pollutants / Q000378:metabolism
D010455:Peptides; D010472:Perchlorates; D014873:Water Pollutants; D010088:Oxidoreductases