Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa.
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With purified subunits the enzyme strongly prefers ribonucleoside triphosphates: CTP gives more than 60-fold higher activity than CDP.
"The results revealed that CTP is the preferred substrate with a >60-fold higher activity compared to CDP"
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The catalytic machinery, the adenosylcobalamin site and the substrate and effector sites are in NrdJa; NrdJb provides the thiol-disulfide exchange function via redox-active C-terminal cysteines.
"the full catalytic machinery for NTP reduction, including AdoCbl cofactor binding, resides within the NrdJa subunit, and that the main role of NrdJb is to provide thiol-disulfide exchange"
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NrdJa is 734 residues and NrdJb 229 residues in P. aeruginosa; the nrdJ gene is split at a specific site in these proteobacteria.
"The nrdJ gene in these species is split at a specific site into two genes, nrdJa and nrdJb, of a single operon that encodes the two polypeptides NrdJa (734 residues in PA) and NrdJb (229 residues in PA)"
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The standard assay used DTT as reductant with AdoCbl, ATP and CTP, giving a specific activity of 1.5 nmol/min/mg; thioredoxin was not used.
"the specific activity of the enzyme was determined to be 1.5 nmol/min/mg (with respect to the NrdJa subunit)"