Enzymatic Activity and Thermodynamic Stability of Biliverdin IXβ Reductase Are Maintained by an Active Site Serine

Abstract

Biliverdin reductase IXβ (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 applied experimental and thermodynamic modeling studies to provide further insight into 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), while 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 the structural basis for the “encounter” and “equilibrium” states of the binary complex which are regulated by NAD(P)H.

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding

“…5c). This is consistent with previous kinetics studies that also indicated a weaker coenzyme affinity [15]. The difference in coenzyme binding affinity would not be expected to significantly affect substrate turnover, which is collected at concentrations significantly higher than the K D of either the wild type or R78A mutant.…”

“…For example, some of the largest CSPs induced by the coenzyme include residues 72–87 that are immediately within the BLVRB active site and the adjacent region of residues 34–39. This is consistent with a recent study that has shown that Arg35 is at least partially responsible for the selectivity of NADPH over NADH, as its side chain forms a direct interaction with the phosphate of the coenzyme [15]. Although in the absence of an apo BLVRB structure, it is difficult to deduce whether CSPs due to coenzyme binding are localized to side chain rearrangements or larger secondary structure rearrangements, it is worth noting that coenzyme binding to BLVRB induces higher CSPs than those previously reported for coenzyme binding to DHFR [23].…”

In silico and crystallographic studies identify key structural features of biliverdin IXβ reductase inhibitors having nanomolar potency

“…Attempts to obtain an experimental structure of the BLVRB-NADP ϩ -NSC130813 complex have been hampered by the low aqueous solubility of the ligand, although crystal structures of the xanthene-based inhibitors erythrosin extra bluish and phloxine B were solvable. Structures were nearly identical to ligand-free BLVRB (22), with ligands found within the ample BLVRB-binding site adjacent to the nicotinamide moiety of the cofactor and bordered by the flexible loops 80, 120, and the N-terminal half of the helix ␣E (27). Although erythrosin extra bluish and phloxine B bind less deeply than FMN or lumichrome into the BLVRB active site, both compounds interact with the side chain of Ser-111, a residue previously identified as critical for maintenance of the enzymatic active site (27) and cellular reductase functions that maintain redox balance and antioxidant handling in hematopoietic cells (26).…”

“…Structures were nearly identical to ligand-free BLVRB (22), with ligands found within the ample BLVRB-binding site adjacent to the nicotinamide moiety of the cofactor and bordered by the flexible loops 80, 120, and the N-terminal half of the helix ␣E (27). Although erythrosin extra bluish and phloxine B bind less deeply than FMN or lumichrome into the BLVRB active site, both compounds interact with the side chain of Ser-111, a residue previously identified as critical for maintenance of the enzymatic active site (27) and cellular reductase functions that maintain redox balance and antioxidant handling in hematopoietic cells (26). Consistent with what is observed in the crystal structures for phloxine B-and erythrosin-bound BLVRB, the halogen located on the tricyclic hydrocarbon of NSC130813 and NSC12516 is predicted to be in close proximity to Ser-111 (Fig.…”

“…Interestingly, recent data applying a genetic screen in humans have identified a BLVRB loss-of-function redox mutation (BLVRB S111L ) that unmasks a fundamentally important mechanism linking heme degradation to the development of exaggerated thrombopoiesis (platelet production) (26). This mutation affects both flavin and biliverdin coupling, and subsequent experimental and thermodynamic studies provided the foundation for the "encounter" and "equilibrium" states of the binary BLVRB-NADP ϩ complex, and the critical role of the active site Ser-111 in maintaining enzymatic function and thermodynamic stability mediated by the hydrogen-bonded network (27). We have now extended these data by integrating molecular modeling and crystallographic studies to identify an initial class of BLVRB small molecule inhibitors displaying nanomolar activity.…”