Dataset Information

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

ABSTRACT: 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.

SUBMITTER: Paukovich N

PROVIDER: S-EPMC6431292 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

Paukovich Natasia N Xue Mengjun M Elder James R JR Redzic Jasmina S JS Blue Ashley A Pike Hamish H Miller Brian G BG Pitts Todd M TM Pollock David D DD Hansen Kirk K D'Alessandro Angelo A Eisenmesser Elan Zohar EZ

Journal of molecular biology 20180620 18 Pt B

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 isothe ...[more]

PMID: 29932944

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Project description:Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F(430). Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F(430) nickel in the deepest part of the 30 A long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 A predominantly hydrophobic part of the channel close to F(430), with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 A. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB(5)SH, CoB(6)SH, CoB(8)SH, and CoB(9)SH, respectively). The data presented here reveal that the shorter CoB(5)SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB(6)SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB(6)SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB(8)SH and CoB(9)SH analogues can be accommodated in the active site through exclusion of this water. These analogues react with Ni(III)-methyl, a proposed MCR catalytic intermediate of methanogenesis. The CoB(8)SH thiolate is 2.6 A closer to the nickel than that of CoBSH, but the additional carbon of CoB(9)SH only decreases the nickel thiolate distance a further 0.3 A. Although the analogues do not induce any structural changes in the substrate channel, the thiolates appear to preferentially bind at two distinct positions in the channel, one being the previously observed CoBSH thiolate position and the other being at a hydrophobic annulus of residues that lines the channel proximal to the nickel.

Dataset Information

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

Publications

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

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