Cooperative Substrate Binding Controls Catalysis in Bacterial Cytochrome P450terp (CYP108A1)

Abstract

Despite being one of the most well-studied aspects of cytochrome P450 chemistry, important questions remain regarding the nature and ubiquity of allosteric regulation of catalysis. The crystal structure of a bacterial P450, P450terp, in the presence of substrate reveals two binding sites, one above the heme in position for regioselective hydroxylation and another in the substrate access channel. Unlike many bacterial P450s, P450terp does not exhibit an open to closed conformational change when substrate binds; instead, P450terp uses the second substrate molecule to hold the first substrate molecule in position for catalysis. Spectral titrations clearly show that substrate binding to P450terp is cooperative with a Hill coefficient of 1.4 and is supported by isothermal titration calorimetry. The importance of the allosteric site was explored by a series of mutations that weaken the second site and that help hold the first substrate in position for proper catalysis. We further measured the coupling efficiency of both the wild-type (WT) enzyme and the mutant enzymes. While the WT enzyme exhibits 97% efficiency, each of the variants showed lower catalytic efficiency. Additionally, the variants show decreased spin shifts upon binding of substrate. These results are the first clear example of positive homotropic allostery in a class 1 bacterial P450 with its natural substrate. Combined with our recent results from P450cam showing complex substrate allostery and conformational dynamics, our present study with P450terp indicates that bacterial P450s may not be as simple as once thought and share complex substrate binding properties usually associated with only mammalian P450s.

3site Multisubstrate-Bound State of Cytochrome P450cam

“…Specifically, binding modes closely resembling hereby the described waiting mode has been crystallographically observed for CYP107 (1EGY.pdb and 1EUP.pdb), CYP158A2 (2D0E.pdb), CYP3A4 (2V0M.pdb and 1W0F.pdb), CYP158A1 (2NZ5.pdb), and CYP2C8 (2NNH.pdb) . More recently, in P450terp, a binding mode, exactly matching the waiting mode (8EUH.pdb), has been claimed during the preparation of this paper . These recurrences underscore the possibility that the 3site state described herein could be a functional reality in numerous P450 isozymes, if not all.…”

“…97 More recently, in P450terp, a binding mode, exactly matching the waiting mode (8EUH.pdb), has been claimed during the preparation of this paper. 98 These recurrences underscore the possibility that the 3site state described herein could be a functional reality in numerous P450 isozymes, if not all.…”

A three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D

“…From substrate point of view, binding of tylosin at the remote allosteric site promotes the binding of the orthosteric tylosin and enlarges the substrate pocket with microscopic conformational changes. And this type of cooperativity with minor conformational change is similar to other examples of allosteric stabilisation 68 . This enriches the profile of the regulatory mechanisms in AKR family, and the NADPH-independent mode of tylosin binding broadens our understanding of AKR substrate loading.…”