Activation of biliverdin-IXα reductase by inorganic phosphate and related anions

Abstract

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.

The effect of pH on the initial rate kinetics of the dimeric biliverdin‐IXα reductase from the cyanobacterium Synechocystis PCC6803

“…Inorganic phosphate anion has been shown to be an activator of human BVR‐A [14]. Increasing amounts of sodium phosphate (0–100 m m ) were added to the sBVR‐A assay using both NADH and NADPH as cofactor and at pH 5 and pH 7.…”

“…In addition to its unique quaternary structure, sBVR‐A also exhibits a sharp pH optimum, which we reproducibly measured as pH 5. This behaviour is in contrast to that displayed by the mammalian BVR‐A monomers, which show activity over a broad range of pH values in the range 5–9 [14]. The cyanobacterial BVR‐A is not subject to the potent substrate inhibition observed with the mammalian forms and this has allowed us to complete a full initial rate study on the Synechocystis enzyme and to rigorously establish that it obeys an ordered steady‐state mechanism.…”

“…In this respect, the enzyme from Synechocystis is of considerable interest because it exhibits a narrow acidic pH optimum compared to the broad range of pH that can support activity for the mammalian enzymes [5,14]. The cyanobacterial enzyme is also refractory to activation by inorganic phosphate when NADH is the cofactor [14]. In preliminary experiments, we observed that sBVR‐A is not subject to the potent substrate inhibition observed with the mammalian enzymes and is therefore the first candidate, among all BVR‐A forms studied to date, where a complete initial rate study can be completed in the absence of a biliverdin‐binding protein as well as at the optimum, presumably physiological, pH.…”

Molecular Modeling to Provide Insight into the Substrate Binding and Catalytic Mechanism of Human Biliverdin-IXα Reductase

“…18 This dual cofactor and dual pH-dependent behavior has been explained by the evidence of a distinct cofactor activation mechanism, in which inorganic phosphate can assist NADH to interact with BVR-A in the same way that the 2’-phosphate of NADPH acts. 19 However, the lack of a clearly defined substrate binding mode has slowed investigations on the catalytic mechanism of this particular enzymatic reduction.…”

Structural and functional features of dimeric dihydrodiol dehydrogenase

“…Residues Arg37, Arg41, His76 and His79 contribute greatly to the high affinity for coenzyme (K m for NADPH/NADP + is 0.56/ 0.55 mM) [46] when compared to a structurally equivalent enzyme such as 1,5-anhydro-D-fructose reduc- [51], demonstrated an increase in activity by the addition of inorganic phosphate and NADH as coenzyme, much like DHDH, [52]. Postulated to take on the role of the 2'-phosphate of NADPH, it is thought to interact with Arg44 (in position and orientation similar to Arg41 in Figure 6.…”