The use of synthetic linear tetrapyrroles to probe the verdin sites of human biliverdin‐IXα reductase and human biliverdin‐IXβ reductase

Abstract

Many vertebrate species express two enzymes that are capable of catalysing the reduction of various isomers of biliverdin. Biliverdin‐IXα reductase (BVR‐A) is most active with its physiological substrate biliverdin‐IXα, but can also reduce the three other biliverdin isomers IXβ, IXδ and IXγ. Biliverdin‐IXβ reductase (BVR‐B) catalyses the reduction of only the IXβ, IXδ and IXγ isomers of biliverdin. Therefore, the activity of BVR‐A can be measured using biliverdin‐IXα as a specific substrate. We now show that the dimethyl esters of biliverdin‐IXβ and biliverdin‐IXδ are substrates for BVR‐B, but not for BVR‐A. This provides a useful method for specifically assaying the activity of both BVR‐A and BVR‐B in crude mixtures, using biliverdin‐IXα for BVR‐A and the dimethyl ester of either biliverdin‐IXβ or biliverdin‐IXδ for BVR‐B. Human BVR‐A has been suggested as a pharmacological target for neonatal jaundice. Because of the absence of a crystal structure with biliverdin bound to BVR‐A, we have investigated indirect ways of examining tetrapyrrole binding. In the present study, we report that a number of sterically locked conformers of 18‐ethylbiliverdin‐IXα are substrates for human BVR‐A, and discuss the implications for the biliverdin binding site. The oxidation of bilirubin‐IXα ditaurate to biliverdin‐IXα ditaurate is also described. We show that biliverdin‐IXα ditaurate is a substrate for human BVR‐A and discuss the possibility of using a competing substrate, which is reduced to a water soluble and excretable rubin, as a prototypic inhibitor of BVR‐A.

Lack of p53 Decreases Basal Oxidative Stress Levels in the Brain Through Upregulation of Thioredoxin-1, Biliverdin Reductase-A, Manganese Superoxide Dismutase, and Nuclear Factor Kappa-B

“…BVR-A, the main isoform of BVR (26,36), catalyzes the reduction of BV into BR and is coexpressed with HO-1 in cells of the rat brain that express these enzymes under normal conditions (23). Furthermore, BVR-A is also a serine/threonine/tyrosine kinase that interacts with members of the mitogen-activated protein kinase family, in particular, the extracellular signalregulated kinases 1/2, and once translocated into the nucleus regulates the expression of oxidative stress-responsive genes…”

Tat-biliverdin reductase A inhibits inflammatory response by regulation of MAPK and NF-κB pathways in Raw 264.7 cells and edema mouse model

“…Humans have two biliverdin isozymes biliverdin reductase A (BLVRA) and biliverdin reductase B (BLVRB). Although both isozymes are related in conversion, BLVRA converts into bilirubin, a more potent antioxidant (Beale and Cornejo, 1984;Franklin et al, 2009). Recent studies have further suggested a role in ROS Extracts from the cells prepared and analyzed for MAP kinase protein activation by Western blot analysis and band intensity by densitometer.…”

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans

“…28 BLVRB was readily detectable in healthy control gel-filtered human platelets with no evidence for altered expression in either BLVRB S111L ET or RT platelets ( Figure 4A ), 30 and uniquely positioned for recognition and/or proton transfer between flavin isoalloxazine and nicotinamide rings. 15 Bacterially expressed and purified recombinant BLVRB WT (wild type) and BLVRB S111L demonstrated disparate NAD(P)H-dependent redox coupling using both flavin-and BV-specific substrates ( Figure 4C-E), the latter uniquely generated by coupled heme oxidation as verdinrestricted BLVRB activity probes retaining no BLVRA cross-reactivity. 15 BLVRB S111L enzymatic activity was defective using flavin mononucleotide (flavin reductase activity; P , .0001) and BV IXb dimethyl esters (BV reductase [BVR] activity; P , .0001), establishing that the S 111 L substitution represents a loss-of-function redox mutation with either substrate.…”

“…15 Bacterially expressed and purified recombinant BLVRB WT (wild type) and BLVRB S111L demonstrated disparate NAD(P)H-dependent redox coupling using both flavin-and BV-specific substrates ( Figure 4C-E), the latter uniquely generated by coupled heme oxidation as verdinrestricted BLVRB activity probes retaining no BLVRA cross-reactivity. 15 BLVRB S111L enzymatic activity was defective using flavin mononucleotide (flavin reductase activity; P , .0001) and BV IXb dimethyl esters (BV reductase [BVR] activity; P , .0001), establishing that the S 111 L substitution represents a loss-of-function redox mutation with either substrate. These results were confirmed in HEK293 cells using lentivirus(Lv)-induced expression, where enhanced BVR activity was evident in HEK293/BLVRB WT -infected cells with little to no activity in HEK293/BLVRB S111L cells compared with HEK293/ Control cells ( Figure 4F); residual BVR activity in HEK293/Control and HEK293/BLVRB S111L reflected endogenous BLVRB expression with no evidence for BLVRA cross-reactivity ( Figure 4G).…”

“…Specific activity determination of BLVRB WT and BLVRB S111L were completed on bacterially expressed recombinant enzymes at 25°C using flavin mononucleotide or pooled biliverdin (BV) dimethyl esters synthesized by coupled oxidation of heme 15 ; RIPA-solubilized cytoplasmic lysates served as the source for cellular BLVRB functional assays, 16 and immunodetection was completed using sheep anti-human BLVRB (R&D Systems; 1:100 dilution) and anti-actin monoclonal antibody (mAb) (EMD Millipore; 1:1000).…”