title: 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 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.
authors.affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, P. R. China.
authors.forename: Wen-Ting
authors.initials: WT
authors.lastname: Chu
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
authors.forename: Natasha M
authors.initials: NM
authors.lastname: Nesbitt
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Gnatenko
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
authors.forename: Zongdong
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, 11794, USA.
authors.forename: Beibei
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, 11794, USA.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Seeliger
authors.affiliation: Department of Biochemistry, Trinity College, Dublin 2, Ireland.
authors.forename: Seamus
authors.initials: S
authors.lastname: Browne
authors.affiliation: Department of Biochemistry, Trinity College, Dublin 2, Ireland.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Mantle
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
authors.forename: Wadie F
authors.initials: WF
authors.lastname: Bahou
authors.affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, P. R. China.|Department of Chemistry and Physics, Stony Brook University, Stony Brook, NY, 11794, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D009243:NAD; D012694:Serine; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase
keywords: cofactors; conformation analysis; enzyme catalysis; molecular dynamics; molecular modeling