Human biliverdin IX alpha reductase is a zinc-metalloprotein - Characterization of purified and Escherichia coli expressed enzymes

Biliverdin IX alpha reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin. BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH: NADPH) specificity. A cDNA clone encoding human BVR was isolated from a lambda library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA. This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed. The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein. The cDNA hybridized with a single message of approximate to 1.2 kb in human kidney poly(A)-rich RNA, and appeared, by Southern blot analysis, to be the product of a single-copy gene. Sequence analysis indicated that the human reductase shows approximate to 83% identity, at both the nucleotide and amino acid levels, with rat BVR. In some regions including the carboxyl terminus, protein sequence identity drops to 45%. Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat). The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions. Western blot analysis and isoelectric focusing demonstrate that: although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio. That human BVR is a Zn metalloprotein was further substantiated by Zn-65 exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent, activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity.