Background:ALDH6A1,a member of the ALDH family,plays a crucial role in the catabolic pathways of valine and thymine.Dysregulation of ALDH6A1 expression has been linked to a variety of diseases.Methylmalonate semialdeh...Background:ALDH6A1,a member of the ALDH family,plays a crucial role in the catabolic pathways of valine and thymine.Dysregulation of ALDH6A1 expression has been linked to a variety of diseases.Methylmalonate semialdehyde dehydrogenase deficiency(MMSDH deficiency),an autosomal recessive disorder,arises from muta-tions in the ALDH6A1 gene.Additionally,ALDH6A1 has emerged as a biomarker for several types of severe cancer.Despite its significance,the structural and biochemical mechanisms of ALDH6A1 remain poorly explored.Methods:The apo form of ALDH6A1 was solved by cryo-electron microscopy.Enzyme activity assay and thermal stability assays were conducted to elucidate the bio-chemical properties of ALDH6A1 and to find an agonist of ALDH6A1,Alda-1.The binding pattern of ALDH6A1 and nicotinamide adenine dinucleotide(NAD^(+))was explored by molecular docking.Results:This study presents,for the first time,a structural analysis of ALDH6A1 in its apo form at a resolution of 2.75Å,uncovering a tetrameric architecture with tightly interacting monomers.Our findings indicate that Alda-1,an agonist of ALDH2,enhances ALDH6A1 activity as well.Moreover,ALDH6A1,compared with ALDH2,exhibits a unique binding model with NAD^(+).Conclusion:Our results shed light on the structural aspects of ALDH6A1 and provide valuable insights into its catalytic mechanism.The precise determination of the ALDH6A1 structure holds promise for the development of targeted therapies aimed at restoring ALDH6A1 activity,thus providing potential value for individuals affected by related diseases.展开更多
Background: Methylmalonate semialdehyde dehydrogenase(ALDH6A1), encoded by the ALDH6A1 gene, is essential for the metabolic degradation of valine and thymine.Genetic alterations in ALDH6A1 lead to methylmalonate semia...Background: Methylmalonate semialdehyde dehydrogenase(ALDH6A1), encoded by the ALDH6A1 gene, is essential for the metabolic degradation of valine and thymine.Genetic alterations in ALDH6A1 lead to methylmalonate semialdehyde dehydrogenase deficiency(MMSDD), a rare disease with only five reported disease mutations, focusing on its molecular foundation but lacking in-depth mechanistic investigations. Therefore, the structural and biochemical properties of the ALDH6A1mutants have not yet been thoroughly examined.Methods: Wild-type(WT) and mutant ALDH6A1 were constructed as plasmids and purified after prokaryotic expression to obtain conformationally homogeneous and pure protein. The structures of ALDH6A1 mutants(P62S, Y172H & R535C, S262Y,P421S, and G446R) were solved using cryo-electron microscopy. Based on the results of ALDH6A1 WT, enzyme activity and thermal stability experiments of their mutants were performed to explore the ALDH6A1's biochemical characteristics.Results: This study presents a structural analysis of the ALDH6A1 mutants, P62S,Y172H & R535C, S262Y, P421S, and G446R at resolutions of 3.70, 2.92, 3.12, 3.47,and 3.00 Å, respectively. However, the electronic density of ALDH6A1 P421S is poor,and it is difficult to fit into this density. Furthermore, the root-mean-square deviation(r.m.s.d.) of ALDH6A1 WT with these mutants was significant. This study revealed a tetrameric structure with closely interacting monomers, except for ALDH6A1 P62S,which forms a dimer, and is consistent with the principles of the aldehyde dehydrogenase family. Moreover, these disease mutations also affect enzyme activity and thermal stability.Conclusions: Our findings shed light on disease mutations that contribute to the properties of ALDH6A1 and lead to the genesis of MMSDD from structural and biochemical perspectives, which holds promise as a potential theoretical basis for this rare disease.展开更多
基金supported by the National Key Research and Development Program of China(2022YFC2703100 and 2023YFC3605504)the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences(2021-I2M-1003)the National High Level Hospital Clinical Research Funding(2022-PUMCH-D-002 and 2022 PUMCH-B-098).
文摘Background:ALDH6A1,a member of the ALDH family,plays a crucial role in the catabolic pathways of valine and thymine.Dysregulation of ALDH6A1 expression has been linked to a variety of diseases.Methylmalonate semialdehyde dehydrogenase deficiency(MMSDH deficiency),an autosomal recessive disorder,arises from muta-tions in the ALDH6A1 gene.Additionally,ALDH6A1 has emerged as a biomarker for several types of severe cancer.Despite its significance,the structural and biochemical mechanisms of ALDH6A1 remain poorly explored.Methods:The apo form of ALDH6A1 was solved by cryo-electron microscopy.Enzyme activity assay and thermal stability assays were conducted to elucidate the bio-chemical properties of ALDH6A1 and to find an agonist of ALDH6A1,Alda-1.The binding pattern of ALDH6A1 and nicotinamide adenine dinucleotide(NAD^(+))was explored by molecular docking.Results:This study presents,for the first time,a structural analysis of ALDH6A1 in its apo form at a resolution of 2.75Å,uncovering a tetrameric architecture with tightly interacting monomers.Our findings indicate that Alda-1,an agonist of ALDH2,enhances ALDH6A1 activity as well.Moreover,ALDH6A1,compared with ALDH2,exhibits a unique binding model with NAD^(+).Conclusion:Our results shed light on the structural aspects of ALDH6A1 and provide valuable insights into its catalytic mechanism.The precise determination of the ALDH6A1 structure holds promise for the development of targeted therapies aimed at restoring ALDH6A1 activity,thus providing potential value for individuals affected by related diseases.
基金supported by the National Key Research and Development Program of China (2023YFC3605504)the Innovation Fund for Medical Sciences from the Chinese Academy of Medical Sciences (2021-I2M-1-003)the National High-Level Hospital Clinical Research Funding (2022-PUMCH-D-002 and 2022-PUMCH-B-098)
文摘Background: Methylmalonate semialdehyde dehydrogenase(ALDH6A1), encoded by the ALDH6A1 gene, is essential for the metabolic degradation of valine and thymine.Genetic alterations in ALDH6A1 lead to methylmalonate semialdehyde dehydrogenase deficiency(MMSDD), a rare disease with only five reported disease mutations, focusing on its molecular foundation but lacking in-depth mechanistic investigations. Therefore, the structural and biochemical properties of the ALDH6A1mutants have not yet been thoroughly examined.Methods: Wild-type(WT) and mutant ALDH6A1 were constructed as plasmids and purified after prokaryotic expression to obtain conformationally homogeneous and pure protein. The structures of ALDH6A1 mutants(P62S, Y172H & R535C, S262Y,P421S, and G446R) were solved using cryo-electron microscopy. Based on the results of ALDH6A1 WT, enzyme activity and thermal stability experiments of their mutants were performed to explore the ALDH6A1's biochemical characteristics.Results: This study presents a structural analysis of the ALDH6A1 mutants, P62S,Y172H & R535C, S262Y, P421S, and G446R at resolutions of 3.70, 2.92, 3.12, 3.47,and 3.00 Å, respectively. However, the electronic density of ALDH6A1 P421S is poor,and it is difficult to fit into this density. Furthermore, the root-mean-square deviation(r.m.s.d.) of ALDH6A1 WT with these mutants was significant. This study revealed a tetrameric structure with closely interacting monomers, except for ALDH6A1 P62S,which forms a dimer, and is consistent with the principles of the aldehyde dehydrogenase family. Moreover, these disease mutations also affect enzyme activity and thermal stability.Conclusions: Our findings shed light on disease mutations that contribute to the properties of ALDH6A1 and lead to the genesis of MMSDD from structural and biochemical perspectives, which holds promise as a potential theoretical basis for this rare disease.
