Evidence that biliverdin-IX beta reductase and flavin reductase are identical.
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ABSTRACT:
SUBMITTER: Shalloe F
PROVIDER: S-EPMC1217361 | biostudies-other | 1996 Jun
REPOSITORIES: biostudies-other
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Project description:Biliverdin reductase IX? (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1)?elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2)?defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP+ complex; 3)?provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.
| S-EPMC5293602 | biostudies-literature
Project description:The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IXbeta reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a K(d) of 15.8 microM was calculated. Equilibrium fluorescence quenching experiments indicated a K(d) of 0.55 microM, suggesting that an enzyme-NADPH encounter complex (K(d) 15.8 microM) isomerizes to a more stable 'nucleotide-induced' conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with K(m) values of 52 microM, 125 microM and 53 microM, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a K(i) of 76 microM, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIIIalpha, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (K(i) 0.59 microM) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IXbeta reductase has also been shown to exhibit ferric reductase activity, with an apparent K(m) of 2.5 microM for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-alpha isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IXbeta reductase.
| S-EPMC1220769 | biostudies-other
Project description:Human biliverdin-IX? reductase (hBVR-A) catalyzes the conversion of biliverdin-IX? to bilirubin-IX? in the last step of heme degradation and is a key enzyme in regulating a wide range of cellular responses. Though the X-ray structure of hBVR-A is available including cofactor, a crystal structure with a bound substrate would be even more useful as a starting point for protein-structure-based inhibitor design, but none have been reported. The present study employed induced fit docking (IFD) to study the substrate binding modes to hBVR-A of biliverdin-IX? and four analogues. The proposed substrate binding modes were examined further by performing molecular dynamics (MD) simulations followed by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations. The predicted binding free energies for the five biliverdin-IX? analogues match well with the relative potency of their reported experimental binding affinities, supporting that the proposed binding modes are reasonable. Furthermore, the ternary complex structure of hBVR-A binding with biliverdin-IX? and the electron donor cofactor NADPH obtained from MD simulations was exploited to investigate the catalytic mechanism, by calculating the reaction energy profile using the quantum mechanics/molecular mechanics (QM/MM) method. On the basis of our calculations, the energetically preferred pathway consists of an initial protonation of the pyrrolic nitrogen on the biliverdin substrate followed by hydride transfer to yield the reduction product. This conclusion is consistent with a previous mechanistic study on human biliverdin IX? reductase (hBVR-B).
| S-EPMC3505555 | biostudies-literature
Project description:Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool.
| S-EPMC10511041 | biostudies-literature
Project description:Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s-1 Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s-1 The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.
| S-EPMC7642083 | biostudies-literature
Project description:Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78?Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.
| S-EPMC6431292 | biostudies-literature
Project description:Conversion of four isomers of meso-oxyprotohaem IX into the corresponding biliverdin IX was attempted with a reconstituted haem oxygenase system in the presence of NADPH-cytochrome c reductase and NADPH. Only the alpha-isomer of meso-oxyprotohaem IX was converted effectively into biliverdin IX alpha, which was further reduced to bilirubin IX alpha by biliverdin reductase. Only trace amounts of biliverdins IX beta, IX gamma and IX delta were respectively formed from the incubation mixture of the corresponding oxyprotohaemin IX isomers with the complete haem oxygenase system under the same conditions. In a kinetic study, the Km for alpha-meso-oxyprotohaem IX was 3.6 microM, which was 2-fold higher than that for protohaem IX. The maximum velocity (Vmax.) of the conversion of alpha-meso-oxyprotohaem IX into biliverdin IX alpha was twice as fast as that of protohaem IX. These results demonstrate that alpha-meso-oxyprotohaem IX is an intermediate of haem degradation and it was converted stereospecifically into biliverdin IX alpha via verdohaem IX alpha.
| S-EPMC1131782 | biostudies-other
Project description:The cellular response to an inflammatory stressor requires a proinflammatory cellular activation followed by a controlled resolution of the response to restore homeostasis. We hypothesized that biliverdin reductase (BVR) by binding biliverdin (BV) quells the cellular response to endotoxin-induced inflammation through phosphorylation of endothelial nitric oxide synthase (eNOS). The generated NO, in turn, nitrosylates BVR, leading to nuclear translocation where BVR binds to the Toll-like receptor-4 (TLR4) promoter at the Ap-1 sites to block transcription. We show in macrophages that BV-induced eNOS phosphorylation (Ser-1177) and NO production are mediated in part by Ca(2+)/calmodulin-dependent kinase kinase. Furthermore, we show that BVR is S-nitrosylated on one of three cysteines and that this posttranslational modification is required for BVR-mediated signaling. BV-induced nuclear translocation of BVR and inhibition of TLR4 expression is lost in macrophages derived from Enos(-/-) mice. In vivo in mice, BV provides protection from acute liver damage and is dependent on the availability of NO. Collectively, we elucidate a mechanism for BVR in regulating the inflammatory response to endotoxin that requires eNOS-derived NO and TLR4 signaling in macrophages.
| S-EPMC3219137 | biostudies-literature
Project description:Pseudomonas aeruginosa is a versatile opportunistic pathogen requiring iron for its survival and virulence within the host. The ability to switch to heme as an iron source provides an advantage in chronic infection. We have recently shown the extracellular heme metabolites biliverdin IX (BVIX) and/or BVIX positively regulate the heme dependent cell surface signaling cascade. We further investigated the role of BVIX and BVIX in cell signaling utilizing allelic strains lacking a functional HemO (hemOin), or one reengineered to produce BVIX (hemO). Compared to PAO1 both strains show a heme dependent growth defect, decreased swarming and twitching and less robust biofilm formation. Interestingly, the motility and biofilm defects were partially rescued on addition of exogenous BVIX and BVIX. Utilizing LC-MS/MS we performed a comparative proteomics and metabolomics analysis of PAO1 versus the allelic strains in shaking and static conditions. In shaking conditions, the hemO allelic strains showed a significant increase in proteins involved in quorum sensing (QS), phenazine production and chemotaxis.Metabolite profiling further revealed increased levels of PQS and phenazine metabolites. In static conditions we observed a significant repression of chemosensory pathways and Type IV pili (TFP) biogenesis proteins as well as several phosphodiesterases associated with biofilm dispersal. We propose BVIX metabolites function as signaling and chemotactic molecules integrating heme utilization as an iron source into the adaptation of P. aeruginosa from a planktonic to sessile lifestyle.
2024-01-19 | PXD045835 | Pride
Project description:The effect of pH on the initial-rate kinetic behaviour of BVR-A (biliverdin-IXalpha reductase) exhibits an alkaline optimum with NADPH as cofactor, but a neutral optimum with NADH as cofactor. This has been described as dual cofactor and dual pH dependent behaviour; however, no mechanism has been described to explain this phenomenon. We present evidence that the apparent peak of activity observed at neutral pH with phosphate buffer and NADH as cofactor is an anion-dependent activation, where inorganic phosphate apparently mimics the role played by the 2'-phosphate of NADPH in stabilizing the interaction between NADH and the enzyme. The enzymes from mouse, rat and human all exhibit this behaviour. This behaviour is not seen with BVR-A from Xenopus tropicalis or the ancient cyanobacterial enzyme from Synechocystis PCC 6803, which, in addition to being refractory to activation by inorganic phosphate, are also differentiated by an acid pH optimum with both nicotinamide nucleotides.
| S-EPMC1925240 | biostudies-literature