Biochemistry & Cellular and Molecular Biology Department, University of Tennessee, 1311 Cumberland Ave., Knoxville, TN 37996, USA.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, 12801 E 17th Ave., Aurora, CO 80045, USA.
J Biochem. 2020 Aug 1;168(2):191-202. doi: 10.1093/jb/mvaa039.
Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme 'clamps' formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.
胆红素还原酶 B（BLVRB）家族成员是维持细胞氧化还原平衡的重要黄素还原酶，最近的研究发现，BLVRB 本身可以决定细胞命运。然而，与大多数酶不同，BLVRB 家族仍然是一个谜，其活性位点在进化过程中不断变化，结构和功能后果未知。在这里，我们应用了一种多方面的方法，结合 X 射线晶体学、NMR 和动力学方法，阐明了进化过程中不断变化的 BLVRB 活性位点的结构和功能基础。使用一组三种 BLVRB 同工型（人、狐猴和蹄兔）和多种人 BLVRB 突变体，我们的研究揭示了一种新的进化机制，其中由活性位点内两个共进化位置的精氨酸侧链形成的辅酶“夹”有助于减慢辅酶释放（位置 14 和 78）。我们发现，较弱的结合底物进一步减慢了辅酶的释放，导致相对较慢的周转数。然而，由进化或诱变产生的不同 BLVRB 活性位点表现出辅酶释放和底物周转之间令人惊讶的反比关系，这与我们在这里测量的更快的氢化物转移化学步骤无关。总之，我们的研究阐明了进化过程中不断变化的 BLVRB 活性位点的作用，该位点有助于调节辅酶释放，并揭示了辅酶释放与底物周转相关。
