Methylmalonate Semialdehyde Research Articles

Structural and biochemical basis for the pathogenic mutations of methylmalonate semialdehyde dehydrogenase ALDH6A1

BackgroundMethylmalonate 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 ALDH6A1 mutants have not yet been thoroughly examined. MethodsWild-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. ResultsThis 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. ConclusionsOur 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.

Structural and biochemical basis of methylmalonate semialdehyde dehydrogenase ALDH6A1

BackgroundALDH6A1, 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 mutations 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. MethodsThe apo form of ALDH6A1 was solved by cryo-electron microscopy. Enzyme activity assay and thermal stability assays were conducted to elucidate the biochemical 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. ResultsThis study presents, for the first time, a structural analysis of ALDH6A1 in its apo form at a resolution of 2.75 Å, 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+. ConclusionOur 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.

Engineering Escherichia coli to assimilate β-alanine as a major carbon source.

The threat of global plastic waste accumulation has spurred the exploration of plastics derived from biological sources. A well-known example is polyester made of 1,3-propanediol (1,3-PDO). However, there is no known pathway to assimilate 1,3-PDO into the central carbon metabolism, posing a potential challenge to upcycling such plastic wastes. Here, we proposed that the 1,3-PDO assimilation pathway could pass through malonate semialdehyde (MSA) as an intermediate. Since MSA is a toxic aldehyde, β-alanine was chosen as a surrogate substrate in this study to construct the lower part of the proposed pathway. To this end, we successfully engineered E. coli MG1655 to assimilate β-alanine as the major carbon source. β-alanine could be easily converted into MSA using a β-alanine/pyruvate transaminase from Pseudomonas aeruginosa (PaBapt). However, the subsequent step to generate acetyl-CoA from MSA was unknown. After a series of phenotype screenings, adaptive laboratory evolution and transcriptomic analysis, two CoA-acylating MSA dehydrogenases from Vibrio natriegens (VnMmsD), were found to be able to complete the metabolic pathway. Optical density at 600 nm (OD600) of the resulting strain E. coli BA02 could reach 4.5 after 96 h. Two approaches were subsequently used to improve its performance. First, PaBapt and both VnMmsDs were expressed from a single plasmid to mitigate antibiotic stress. Second, a native 3-hydroxy acid dehydrogenase (EcYdfG) was disrupted to address the carbon loss to 3-hydroxypropionate (3-HP) production from MSA. OD600 of the best-performing strain E. coli BA07∆ could reach 6 within 24 h using 5 g/L β-alanine. The construction of E. coli BA07∆ lays a solid foundation to establishing a 1,3-PDO assimilation pathway. KEYPOINTS: • This study demonstrates the implementation of a metabolic pathway to assimilate β-alanine as the major carbon source in E. coli MG1655. • Two V. natriegens CoA-acylating methyl malonate semialdehyde dehydrogenases were used to complete the pathway in E. coli BA02. • The construction of E. coli BA02 also revealed the plasmid fusion event between two plasmids with the same replication origin.

1276-P: Transancestral Genomic Analysis Links Dysregulation of Valine Metabolism to the Development of Heart Failure in Type 2 Diabetes

Persons with type 2 diabetes (T2D) remain at increased risk of heart failure (HF) despite control of known cardiovascular risk factors. The aim of this study was to identify genes and molecular pathways specifically linking T2D to HF. To this end, we conducted a trans-ancestral genome-wide association study (GWAS) using data from ACCORD, ORIGIN, and six TOPMed cohorts (ARIC, CHS, FHS, JHS, MESA, WHI) . Included were a total of 19,8 participants with T2D (11,852 Whites, 4,152 Blacks, 3,157 Hispanics, and 778 Asians) , 1,8 of whom had developed HF, as defined in each study, after being diagnosed with T2D. A novel genome-wide significant trans-ancestral locus was identified on chromosome 7q21 as being associated with HF (lead SNP=rs6462012, OR=1.25[1.15-1.35], p=3.3 × 10-8) . The lead SNP had similar effect in all ethnicities (Whites: OR=1.28[1.16-1.42], p=9.0 × 10-7; Blacks: OR=1.24[1.06-1.42], p=5.0 × 10-3; Asians: OR=2.38[0.66-12.95], p=0.20) , except Hispanics (OR=1.00[0.80-1.26], p=0.98) . This HF locus appeared to be specific to T2D as it was not found among non-diabetic subjects from the six TOPMed cohorts (OR=1.01[0.95-1.08], p=0.71) or in previous GWAS from the general population. Among subjects with T2D included in the GTEx database, the risk allele at this locus was associated with decreased myocardial expression (beta=-0.19, SE=0.07, p=0.02) of the nearby HIBADH gene coding for the enzyme converting 3-hydroxyisobutyrate (a valine metabolite) to methylmalonic acid semialdehyde, while there was no such association among non-diabetic subjects (beta=0.009, SE=0.03, p=0.77) . In conclusion, we have identified a new HF locus specific for T2D that is linked to branched chain amino acid metabolism, the dysregulation of which has been previously implicated in the development of HF. Further investigation of the mechanisms linking this locus to HF may foster new HF-preventing interventions specifically targeted to the T2D population. Disclosure Y.Tang: None. J.I.Rotter: None. S.S.Rich: None. J.B.Meigs: Consultant; Quest Diagnostics. A.Manning: None. J.Mychaleckyj: None. A.Doria: Research Support; Novo Nordisk Foundation. C.Nlbi trans-omics for precision medicine (topmed) : n/a. M.Pigeyre: n/a. X.Sun: n/a. M.Morieri: Advisory Panel; Merck Sharp & Dohme Corp., Consultant; neopharmed gentili, SLAPharma, Other Relationship; Lilly, Novo Nordisk, Servier Laboratories. H.Shah: None. G.Pare: Advisory Panel; Amgen Inc., Bayer AG, Sanofi, Research Support; Bayer AG. H.C.Gerstein: Advisory Panel; Abbott, Eli Lilly and Company, Hanmi Pharm. Co., Ltd., Novo Nordisk, Pfizer Inc., Sanofi, Viatris Inc., Consultant; Kowa Company, Ltd., Other Relationship; DKSH, Eli Lilly and Company, Sanofi, Zuellig Pharma Holdings Pte. Ltd., Research Support; AstraZeneca, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk, Sanofi. K.Taylor: None. V.S.Ramachandran: None. Funding NIH Biodata Catalyst Fellowship (1OT3HL142479-01, 1OT3HL142478-01, 1OT3HL142481-01, 1OT3HL142480-01, 1OT3HL147154) .

3-Hydroxyisobutyric acid dehydrogenase deficiency: Expanding the clinical spectrum and quantitation of D- and L-3-Hydroxyisobutyric acid by an LC-MS/MS method.

A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients.

Identification of ALDH6A1 as a Potential Molecular Signature in Hepatocellular Carcinoma via Quantitative Profiling of the Mitochondrial Proteome.

Various liver diseases, including hepatocellular carcinoma (HCC), have been linked to mitochondrial dysfunction, reduction of reactive oxygen species (ROS), and elevation of nitric oxide (NO). In this study, we subjected the human liver mitochondrial proteome to extensive quantitative proteomic profiling analysis and molecular characterization to identify potential signatures indicative of cancer cell growth and progression. Sequential proteomic analysis identified 2452 mitochondrial proteins, of which 1464 and 2010 were classified as nontumor and tumor (HCC) mitochondrial proteins, respectively, with 1022 overlaps. Further metabolic mapping of the HCC mitochondrial proteins narrowed our biological characterization to four proteins, namely, ALDH4A1, LRPPRC, ATP5C1, and ALDH6A1. The latter protein, a mitochondrial methylmalonate semialdehyde dehydrogenase (ALDH6A1), was most strongly suppressed in HCC tumor regions (∼10-fold decrease) in contrast to LRPPRC (∼6-fold increase) and was predicted to be present in plasma. Accordingly, we selected ALDH6A1 for functional analysis and engineered Hep3B cells to overexpress this protein, called ALDH6A1-O/E cells. Since ALDH6A1 is predicted to be involved in mitochondrial respiration, we assessed changes in the levels of NO and ROS in the overexpressed cell lines. Surprisingly, in ALDH6A1-O/E cells, NO was decreased nearly 50% but ROS was increased at a similar level, while the former was restored by treatment with S-nitroso-N-acetyl-penicillamine. The lactate levels were also decreased relative to control cells. Propidium iodide and Rhodamine-123 staining suggested that the decrease in NO and increase in ROS in ALDH6A1-O/E cells could be caused by depolarization of the mitochondrial membrane potential (ΔΨ). Taken together, our results suggest that hepatic neoplastic transformation appears to suppress the expression of ALDH6A1, which is accompanied by a respective increase and decrease in NO and ROS in cancer cells. Given the close link between ALDH6A1 suppression and abnormal cancer cell growth, this protein may serve as a potential molecular signature or biomarker of hepatocarcinogenesis and treatment responses.

Clinical, biochemical, mitochondrial, and metabolomic aspects of methylmalonate semialdehyde dehydrogenase deficiency: Report of a fifth case

Methylmalonate semialdehyde dehydrogenase deficiency (MMSDD; MIM 614105) is a rare autosomal recessive defect of valine and pyrimidine catabolism. Four prior MMSDD cases are published. We present a fifth case, along with functional and metabolomic analysis. The patient, born to non-consanguineous parents of East African origin, was admitted at two weeks of age for failure to thrive. She was nondysmorphic, had a normal brain MRI, and showed mild hypotonia. Gastroesophageal reflux occurred with feeding. Urine organic acid assessment identified excess 3-hydroxyisobutyrate and 3-hydroxypropionate, while urine amino acid analysis identified elevated concentrations of β-aminoisobutyrate and β-alanine. Plasma amino acids showed an elevated concentration of β-aminoisobutyrate with undetectable β-alanine. ALDH6A1 gene sequencing identified a homozygous variant of uncertain significance, c.1261C > T (p.Pro421Ser). Management with valine restriction led to reduced concentration of abnormal analytes in blood and urine, improved growth, and reduced gastroesophageal reflux. Western blotting of patient fibroblast extracts demonstrated a large reduction of methylmalonate semialdehyde dehydrogenase (MMSD) protein. Patient cells displayed compromised mitochondrial function with increased superoxide production, reduced oxygen consumption, and reduced ATP production. Metabolomic profiles from patient fibroblasts demonstrated over-representation of fatty acids and fatty acylcarnitines, presumably due to methylmalonate semialdehyde shunting to β-alanine and subsequently to malonyl-CoA with ensuing increase of fatty acid synthesis. Previously reported cases of MMSDD have shown variable clinical presentation. Our case continues the trend as clinical phenotypes diverge from prior cases. Recognition of mitochondrial dysfunction and novel metabolites in this patient provide the opportunity to assess future patients for secondary changes that may influence clinical outcome.

Glomerular Proteomic Profiles in the NZB/W F1 Hybrid Mouse Model of Lupus Nephritis.

BackgroundLupus nephritis is one of the most serious complications of systemic lupus erythematosus (SLE) and is associated with patient mortality. This study aimed to investigate the proteomic profiles of the glomerulus in the NZB/W F1 hybrid mouse model of mild and severe lupus nephritis using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS).Material/MethodsFemale NZB/WF1 mice (n=60) at 28 weeks of age were divided into the mild proteinuria group (+1), the moderate proteinuria group (+2), and the severe proteinuria group (+3) using paper strip urine testing, and then later divided into a mild (≤1+) and severe (≥3+) proteinuria group to allow comparison of upregulation and down-regulation of proteins between the two groups. Renal glomeruli were isolated following renal perfusion with magnetic beads. Protein expression was determined by Western blot, immunohistochemistry, 2D-DIGE, and MALDI-TOF-MS.ResultsA total of 56 differentially expressed proteins were identified from 133 protein spots, of which 18 were upregulated and 23 were down-regulated between groups 1 and 2. Expression of the proteins Ras-related GTP-binding protein B (RRAGB), serine/threonine-protein kinase 1 (SMG1), angiopoietin 2 (ANGP2), methylmalonate semialdehyde (MMSA), and ATP beta chain (ATPB) were identified by Western blot and SMG1, ANGP2, and MMSA were identified by immunohistochemistry.ConclusionsIn a mouse model of lupus nephritis, expression of SMG1, MMSA, and ATPB were down-regulated, and RRAGB and ANGP2 were upregulated.

Disruptions in valine degradation affect seed development and germination in Arabidopsis

We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched-chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3-hydroxyisobutyryl-CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched-chain amino acid catabolism plays in seed development and amino acid homeostasis.

Kinetic Characterization of 3‐Hydroxyisobutyrate Dehydrogenase from Arabidopsis thaliana

Branched‐chain amino acids (BCAAs) initially share a similar metabolic pathway. However, their paths diverge, resulting in unique catabolic pathways for each BCAA. This lab examined valine degradation, as it is the least characterized of all the BCAAs. During valine metabolism, the enzyme 3‐hydroxyisobutyrate dehydrogenase (HIBDH) catalyzes 3‐hydroxyisobutyric acid into methylmalonic semialdehyde as one of the concluding reactions. While HIBDH from Pseudomonas denitrificans had previously been cloned and expressed in Escherichia coli, the goal of this research was to examine the activity of HIBDH in plants. Thus, we cloned the HIBDH gene from Arabidopsis thaliana into E.coli, where its activity could be examined. Two HIBDH constructs were created in order to determine proper exclusion of the mitochondrial leader sequence; these constructs differ only by the length of their amino acid sequences. The expressed proteins were then purified by affinity chromatography and kinetically characterized. The construct composed of nucleotides 97–1044 (denoted At316) resulted in ~9 mg/mL of purified protein, while the construct generated from nucleotides 115–1044 (denoted At310) produced ~1 mg/mL from a similar volume and density of cells at the point of induction. Both constructs resulted in active protein using S‐HIBA (native substrate) and NAD, although very little activity was seen using construct At310. Construct At316 functioned optimally under alkaline conditions similar to HIBDH from P. denitrificans and Homo sapiens. Overall, the biochemical properties of HIBDH in A. thaliana were assessed, offering new insights into both the valine degradation pathway as a whole, as well as the biological and chemical parameters necessary for HIBDH function in plants.Support or Funding InformationThis project was funded by Kenyon College.

Crystal structure and modeling of the tetrahedral intermediate state of methylmalonate-semialdehyde dehydrogenase (MMSDH) from Oceanimonas doudoroffii.

The gene product of dddC (Uniprot code G5CZI2), from the Gram-negative marine bacterium Oceanimonas doudoroffii, is a methylmalonate-semialdehyde dehydrogenase (OdoMMSDH) enzyme. MMSDH is a member of the aldehyde dehydrogenase superfamily, and it catalyzes the NAD-dependent decarboxylation of methylmalonate semialdehyde to propionyl-CoA. We determined the crystal structure of OdoMMSDH at 2.9 Å resolution. Among the twelve molecules in the asymmetric unit, six subunits complexed with NAD, which was carried along the protein purification steps. OdoMMSDH exists as a stable homodimer in solution; each subunit consists of three distinct domains: an NAD-binding domain, a catalytic domain, and an oligomerization domain. Computational modeling studies of the OdoMMSDH structure revealed key residues important for substrate recognition and tetrahedral intermediate stabilization. Two basic residues (Arg103 and Arg279) and six hydrophobic residues (Phe150, Met153, Val154, Trp157, Met281, and Phe449) were found to be important for tetrahedral intermediate binding. Modeling data also suggested that the backbone amide of Cys280 and the side chain amine of Asn149 function as the oxyanion hole during the enzymatic reaction. Our results provide useful insights into the substrate recognition site residues and catalytic mechanism of OdoMMSDH.

Mycobacterium tuberculosis MmsA, a novel immunostimulatory antigen, induces dendritic cell activation and promotes Th1 cell-type immune responses

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is an outstanding pathogen that modulates the host immune response. This inconvenient truth drives the continual identification of antigens that generate protective immunity, including Th1-type T cell immunity. Here, the contribution of methylmalonate semialdehyde dehydrogenase (MmsA, Rv0753c) of Mtb to immune responses was examined in the context of dendritic cell (DC) activation and T cell immunity both in vitro and in vivo. The results showed that MmsA induced DC activation by activating the MAPK and NF-κB signaling pathways. Additionally, MmsA-treated DCs activated naïve T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and IL-2, and induced T cell proliferation. These results indicate that MmsA is a novel DC maturation-inducing antigen that drives the Th1 immune response. Thus, MmsA was found to potentially regulate immune responses via DC activation toward Th1-type T cell immunity, enhancing our understanding of Mtb pathogenesis.

Proteomic analysis of homocholine catabolic pathway in Pseudomonas sp. strain A9

Homocholine, a quaternary ammonium compound that resembles choline in many cholinergic metabolisms, is recently incorporated in many synthetic pharmaceuticals drugs targeting various types of cancer and bone marrow cells. Here, we used a proteomic approach (two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF-MS) to elucidate profoundly the degradation pathway of homocholine by Pseudomonas sp. strain A9. Eighty-nine differentially abundant proteins were identified and were found to cover a broad range of biological processes and cellular responses of strain A9 to homocholine. Of them, most of the proteins involved in the direct uptake and catabolism of homocholine were identified. These results demonstrated that strain A9 uptake homocholine from the media with different transporting systems and then consequently catabolize it to acetyl-CoA and trimethylamine (TMA) by five enzymes namely homocholine dehydrogenase, β-alaninebetaine aldehyde dehydrogenase, β-alaninebetaine CoA transferase, 3-hydroxypropionate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. The acetyl-CoA enters the TCA and glyoxylate pathways to generate energy and provide metabolic precursors for other pathways while TMA exclude out of the bacterial cells raising some environmental and health concerns. Overall, the findings of this study provide a first insight into the global proteomic changes occurring in the strain A9 cells under exposure to homocholine in the environment.

Unraveling the function of paralogs of the aldehyde dehydrogenase super family from Sulfolobus solfataricus

Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles, e.g., in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus, five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and α-ketoglutaric semialdehyde dehydrogenase (α-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)(+)-dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD(+), SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)(+)). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and α-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in γ-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO-SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218, methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD(+)- and CoA-dependent oxidation of methylmalonate semialdehyde, malonate semialdehyde as well as propionaldehyde (PA). For MSDH, a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.

Mutations in ALDH6A1 encoding methylmalonate semialdehyde dehydrogenase are associated with dysmyelination and transient methylmalonic aciduria

BackgroundMethylmalonate semialdehyde dehydrogenase (MMSDH) deficiency is a rare autosomal recessive disorder with varied metabolite abnormalities, including accumulation of 3-hydroxyisobutyric, 3-hydroxypropionic, 3-aminoisobutyric and methylmalonic acids, as well as β-alanine. Existing reports describe a highly variable clinical and biochemical phenotype, which can make diagnosis a challenge. To date, only three reported cases have been confirmed at the molecular level, through identification of homozygous mutations in ALDH6A1, the gene encoding MMSDH. Confirmation by enzyme assay has until now not been possible, due to the extreme instability of the enzyme substrate.Methods and resultsWe report a child with severe developmental delays, abnormal myelination on brain MRI, and transient/variable elevations in lactate, methylmalonic acid, 3-hydroxyisobutyric and 3-aminoisobutyric acids. Compound heterozygous mutations were identified by exome sequencing and confirmed by Sanger sequencing within exon 6 (c.514 T > C; p. Tyr172His) and exon 12 (c.1603C > T; p. Arg535Cys) of ALDH6A1. The resulting amino acid changes, both occurring in residues conserved among mammals, are predicted to be damaging at the protein level. Subsequent MMSDH enzyme assay demonstrated reduced activity in patient fibroblasts, measuring 2.5 standard deviations below the mean.ConclusionsWe present the fourth reported case of MMSDH deficiency with confirmation at the molecular level, and expand on what is already an extremely variable clinical and biochemical phenotype. Furthermore, this is the first report to demonstrate a corresponding reduction in MMSDH enzyme activity. This report illustrates the emerging utilization of whole exome sequencing and variant data filtering using clinical data as an early tool in the diagnosis of rare and variable conditions.

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.

Comparative proteomic analysis of early salt stress-responsive proteins in roots of SnRK2 transgenic rice

BackgroundThe rice roots are highly salt-sensitive organ and primary root growth is rapidly suppressed by salt stress. Sucrose nonfermenting 1-related protein kinase2 (SnRK2) family is one of the key regulator of hyper-osmotic stress signalling in various plant cells. To understand early salt response of rice roots and identify SnRK2 signaling components, proteome changes of transgenic rice roots over-expressing OSRK1, a rice SnRK2 kinase were investigated.ResultsProteomes were analyzed by two-dimensional electrophoresis and protein spots were identified by LC-MS/MS from wild type and OSRK1 transgenic rice roots exposed to 150 mM NaCl for either 3 h or 7 h. Fifty two early salt -responsive protein spots were identified from wild type rice roots. The major up-regulated proteins were enzymes related to energy regulation, amino acid metabolism, methylglyoxal detoxification, redox regulation and protein turnover. It is noted that enzymes known to be involved in GA-induced root growth such as fructose bisphosphate aldolase and methylmalonate semialdehyde dehydrogenase were clearly down-regulated. In contrast to wild type rice roots, only a few proteins were changed by salt stress in OSRK1 transgenic rice roots. A comparative quantitative analysis of the proteome level indicated that forty three early salt-responsive proteins were magnified in transgenic rice roots at unstressed condition. These proteins contain single or multiple potential SnRK2 recognition motives. In vitro kinase assay revealed that one of the identified proteome, calreticulin is a good substrate of OSRK1.ConclusionsOur present data implicate that rice roots rapidly changed broad spectrum of energy metabolism upon challenging salt stress, and suppression of GA signaling by salt stress may be responsible for the rapid arrest of root growth and development. The broad spectrum of functional categories of proteins affected by over-expression of OSRK1 indicates that OSRK1 is an upstream regulator of stress signaling in rice roots. Enzymes involved in glycolysis, branched amino acid catabolism, dnaK-type molecular chaperone, calcium binding protein, Sal T and glyoxalase are potential targets of OSRK1 in rice roots under salt stress that need to be further investigated.

Biochemical and Structural Studies of Uncharacterized Protein PA0743 from Pseudomonas aeruginosa Revealed NAD+-dependent l-Serine Dehydrogenase

The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD(+)-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2-2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD(+) complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.

3‐Hydroxyisobutyrate aciduria and mutations in the ALDH6A1 gene coding for methylmalonate semialdehyde dehydrogenase

3-hydroxyisobutyric aciduria is an organic aciduria with a poorly understood biochemical basis. It has previously been assumed that deficiency of 3-hydroxyisobutyrate dehydrogenase (HIBADH) in the valine catabolic pathway is the underlying enzyme defect, but more recent evidence makes it likely that individuals with 3-hydroxyisobutyryic aciduria represent a heterogeneous group with different underlying mechanisms, including respiratory chain defects or deficiency of methylmalonate semialdehyde dehydrogenase. However, to date methylmalonate semialdehyde dehydrogenase deficiency has only been demonstrated at the gene level for a single individual. We present two unrelated patients who presented with developmental delay and increased urinary concentrations of 3-hydroxyisobutyric acid. Both children were products of consanguineous unions and were of European or Pakistani descent. One patient developed a febrile illness and subsequently died from a hepatoencephalopathy at 2 years of age. Further studies were initiated and included tests of the HIBADH enzyme in fibroblast homogenates, which yielded normal activities. Sequencing of the ALDH6A1 gene (encoding methylmalonate semialdehyde dehydrogenase) suggested homozygosity for the missense mutation c.785 C > A (S262Y) in exon 7 which was not found in 210 control alleles. Mutation analysis of the ALDH6A1 gene of the second patient confirmed the presence of a different missense mutation, c.184 C > T (P62S), which was also identified in 1/530 control chromosomes. Both mutations affect highly evolutionarily conserved amino acids of the methylmalonate semialdehyde dehydrogenase protein. Mutation analysis in the ALDH6A1 gene can reveal a cause of 3-hydroxyisobutyric aciduria, which may present with only slightly increased urinary levels of 3-hydroxyisobutyric acid, if a patient is metabolically stable.

The RpiR-Like Repressor IolR Regulates Inositol Catabolism in Sinorhizobium meliloti

Sinorhizobium meliloti, the nitrogen-fixing symbiont of alfalfa, has the ability to catabolize myo-, scyllo-, and D-chiro-inositol. Functional inositol catabolism (iol) genes are required for growth on these inositol isomers, and they play a role during plant-bacterium interactions. The inositol catabolism genes comprise the chromosomally encoded iolA (mmsA) and the iolY(smc01163)RCDEB genes, as well as the idhA gene located on the pSymB plasmid. Reverse transcriptase assays showed that the iolYRCDEB genes are transcribed as one operon. The iol genes were weakly expressed without induction, but their expression was strongly induced by myo-inositol. The putative transcriptional regulator of the iol genes, IolR, belongs to the RpiR-like repressor family. Electrophoretic mobility shift assays demonstrated that IolR recognized a conserved palindromic sequence (5'-GGAA-N6-TTCC-3') in the upstream regions of the idhA, iolY, iolR, and iolC genes. Complementation assays found IolR to be required for the repression of its own gene and for the downregulation of the idhA-encoded myo-inositol dehydrogenase activity in the presence and absence of inositol. Further expression studies indicated that the late pathway intermediate 2-keto-5-deoxy-D-gluconic acid 6-phosphate (KDGP) functions as the true inducer of the iol genes. The iolA (mmsA) gene encoding methylmalonate semialdehyde dehydrogenase was not regulated by IolR. The S. meliloti iolA (mmsA) gene product seems to be involved in more than only the inositol catabolic pathway, since it was also found to be essential for valine catabolism, supporting its more recent annotation as mmsA.