A collaborative research team led by Zhang Shuyang, President of PUMCH, and Luan Xiaodong, Director of the Drug Development and Evaluation Platform under PUMCH, together with researcher Chen Houzao from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, has made a significant breakthrough in understanding isovaleric acidemia. The team presented the cryo-electron microscopy (cryo-EM) structures of human IVD, resolved in both its apo form and in complex with its substrates, elucidating its catalytic mechanism and pathogenic mutations. This groundbreaking work, published recently in Research (a tier 1 journal, ranked among the top 5% by the Chinese Academy of Sciences, IF=8.5), opens up new therapeutic possibilities for this rare genetic disorder.

IVD plays a crucial role in metabolizing leucine, an essential amino acid. When IVD malfunctions, toxic leucine metabolites accumulate in the body, causing isovaleric acidemia. Children with this condition typically suffer from vomiting, metabolic acidosis, and in severe cases, brain damage. Despite knowing that IVD gene mutations cause the disease, scientists have struggled to precisely decipher the mechanisms behind its functional failures until now, largely due to its highly dynamic structure and complex substrate binding mechanisms.

Using state-of-the-art cryo-EM, the research team captured views of IVD—both in its apo form and in complex with its substrates, isovaleryl-CoA and butyryl-CoA. The images, resolved at near-atomic resolution (2.55?), revealed for the first time that IVD is characterized by a tetrameric architecture composed of four identical subunits, like a "four-leaf clover", with each subunit featuring two α-helical domains and one β-sheet domain arranged in a distinctive "U-shape". This architecture facilitates the enzyme's preference for short branched-chain acyl-CoAs. Importantly, the team discovered that flavin adenine dinucleotide (FAD) serves dual functions—not only participating in catalysis but also bridging monomers like “glue” to facilitate the tetramer assembly.

▲Overall cryo-EM structure of IVD

The research team further replicated clinically common IVD mutations and investigated their impact on IVD's physiological function through biochemical experiments and structural analysis. The research found that mutations such as A314V, S281G, F382V, and E411K severely impair IVD's function, while mutations like R53P, R53C, and A300V have relatively minor effects, allowing IVD to retain partial functionality. Researchers found that mutations that severely affect function mostly disrupt the binding between IVD and FAD, analogous to damaging the core power connection of a machine, preventing IVD from working efficiently. These findings provide atomic-level explanations for the pathogenic mechanisms of mutations as the structural basis for their clinical phenotypic differences.

This research significantly advances our understanding of the structure-function relationship of IVD and opens up promising new approaches for treating isovaleric acidemia, a rare metabolic disorder. The detailed structural information enables researchers to design small molecules that could target either the FAD binding region or substrate pocket, potentially stabilizing mutant forms of IVD and restoring some functionality.

Furthermore, the research established connections between “mutation-structure-function”, which can improve the accuracy of genetic diagnosis of isovaleric acidemia and predict prognosis. For example, patients with milder mutations like A300V might successfully manage their condition through dietary adjustments, while those with severe mutations such as E411K would require early intervention. According to Dr. Luan Xiaodong, these findings lay the groundwork for developing more targeted and effective drugs that could substantially improve outcomes for patients.

Written by Luan Xiaodong and Gan Dingzhu