Cloning and overexpression of rat kidney biliverdin IX alpha reductase as a fusion protein with glutathione S-transferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity.
Native biliverdin IX alpha reductase (BVR-A) is a monomer of molecular mass 34 kDa. We have developed an expression vector that allows the isolation of 40 mg of a glutathione S-transferase (GST)-BVR-A fusion protein from 1 litre of culture. The fusion protein (60 kDa) behaves as a dimer on gel filtration (120 kDa), so that we have artificially created a BVR-A dimer. The recombinant rat kidney enzyme exhibits pre-steady-state 'burst' kinetics that show a pH dependence similar to that already described for ox kidney BVR-A. Similar behaviour was obtained in the presence and absence of the GST domain both for the burst kinetics and during initial-rate studies in the presence and absence of albumin. The stereospecificity of the BVR-A-catalysed oxidation of [4-3H]NADH, labelled at the A and B faces, was shown to occur exclusively via the B face.
Ennis
O
O
Department of Biochemistry, Trinity College, Dublin, Ireland.
Maytum
R
R
Mantle
T J
TJ
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001664:Biliverdine / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013237:Stereoisomerism
D011993:Recombinant Fusion Proteins; D009243:NAD; D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D005969:Glutamate Dehydrogenase; D005982:Glutathione Transferase; D001664:Biliverdine