His74 conservation in the bilin reductase PcyA family reflects an important role in protein-substrate structure and dynamics

• Published in: Archives of biochemistry and biophysics Vol. 537; no. 2; pp. 233 - 242
• Main Authors: Kabasakal, Burak V., Gae, David D., Li, Jie, Lagarias, J. Clark, Koehl, Patrice, Fisher, Andrew J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.09.2013
• Subjects: active sites; Amino Acid Sequence; Bile Pigments - chemistry; Biliverdin; Computer Simulation; Conserved Sequence; Enzyme Activation; enzyme activity; Ferredoxin; histidine; Histidine - chemistry; hydrogen bonding; MD simulations; Models, Chemical; Models, Molecular; molecular dynamics; Molecular Sequence Data; Oxidoreductase; Oxidoreductases - chemistry; Oxidoreductases - ultrastructure; phycobiliprotein; Phycocyanobilin; phytochrome; site-directed mutagenesis; Structure-Activity Relationship; Substrate Specificity; X-ray diffraction; MD simulations; Biliverdin; Ferredoxin; Oxidoreductase; Phycocyanobilin
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2013.07.021

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction of biliverdin IXα’s two vinyl groups to produce phycocyanobilin, an essential chromophore for phytochromes, cyanobacteriochromes and phycobiliproteins. Previous site directed mutagenesis studies indicated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits proton transfer. Here, we exploit X-ray crystallography, enzymology and molecular dynamics simulations to understand the functional role of this invariant histidine. The structures of the H74A, H74E and H74Q variants of PcyA reveal that a “conserved” buried water molecule that bridges His74 and catalytically essential His88 is not required for activity. Despite distinct conformations of Glu74 and Gln74 in the H74E and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggesting smaller residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular dynamic simulations further reveal that the crucial active site residue Asp105 is more dynamic in H74A compared to wild-type PcyA and the two other His74 variants, supporting the conclusion that the Ala74 mutation has increased the flexibility of the active site.