isovaleryl-CoA Dehydrogenase Research Articles

Broad connections in the Arabidopsis seed metabolic network revealed by metabolite profiling of an amino acid catabolism mutant

An Arabidopsis thaliana mutant was identified as having increases in 12 of 20 free proteogenic amino acids in seeds. Because these metabolites are produced from multiple, seemingly unrelated biosynthetic networks, it was not possible to use a candidate gene approach to discover the enzyme defect responsible for this complex syndrome. Complementary metabolite profiling analyses revealed increased seed homomethionine and isovaleroyloxypropyl-glucosinolate, along with reduced 3-benzoyloxypropyl-glucosinolate. These data led to the discovery of impaired branched chain amino acid catabolic enzyme isovaleryl-CoA dehydrogenase (encoded by gene At3g45300 or atIVD) as the cause of this metabolic syndrome. These results indicate that catabolism plays an important role in regulating levels of branched chain amino acids in seeds. The diverse set of metabolites affected in the ivd1 mutants suggests the existence of a more complex network regulating seed amino acid accumulation than previously observed. This combined targeted and non-targeted metabolite profiling approach is broadly applicable to the characterization of metabolic mutants, human disease studies and crop germplasm.

Acyl-CoA Dehydrogenases: Dynamic History of Protein Family Evolution

The acyl-CoA dehydrogenases (ACADs) are enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters in fatty acid and amino acid catabolism. Eleven ACADs are now recognized in the sequenced human genome, and several homologs have been reported from bacteria, fungi, plants, and nematodes. We performed a systematic comparative genomic study, integrating homology searches with methods of phylogenetic reconstruction, to investigate the evolutionary history of this family. Sequence analyses indicate origin of the family in the common ancestor of Archaea, Bacteria, and Eukaryota, illustrating its essential role in the metabolism of early life. At least three ACADs were already present at that time: ancestral glutaryl-CoA dehydrogenase (GCD), isovaleryl-CoA dehydrogenase (IVD), and ACAD10/11. Two gene duplications were unique to the eukaryotic domain: one resulted in the VLCAD and ACAD9 paralogs and another in the ACAD10 and ACAD11 paralogs. The overall patchy distribution of specific ACADs across the tree of life is the result of dynamic evolution that includes numerous rounds of gene duplication and secondary losses, interdomain lateral gene transfer events, alteration of cellular localization, and evolution of novel proteins by domain acquisition. Our finding that eukaryotic ACAD species are more closely related to bacterial ACADs is consistent with endosymbiotic origin of ACADs in eukaryotes and further supported by the localization of all nine previously studied ACADs in mitochondria.

Proteome changes induced by aluminium stress in tomato roots

Growth inhibition in acid soils due to Al stress affects crop production worldwide. To understand mechanisms in sensitive crops that are affected by Al stress, a proteomic analysis of primary tomato root tissue, grown in Al-amended and non-amended liquid cultures, was performed. DIGE-SDS-MALDI-TOF-TOF analysis of these tissues resulted in the identification of 49 proteins that were differentially accumulated. Dehydroascorbate reductase, glutathione reductase, and catalase enzymes associated with antioxidant activities were induced in Al-treated roots. Induced enzyme proteins associated with detoxification were mitochondrial aldehyde dehydrogenase, catechol oxidase, quinone reductase, and lactoylglutathione lyase. The germin-like (oxalate oxidase) proteins, the malate dehydrogenase, wali7 and heavy-metal associated domain-containing proteins were suppressed. VHA-ATP that encodes for the catalytic subunit A of the vacuolar ATP synthase was induced and two ATPase subunit 1 isoforms were suppressed. Several proteins in the active methyl cycle, including SAMS, quercetin 3-O-methyltransferase and AdoHcyase, were induced by Al stress. Other induced proteins were isovaleryl-CoA dehydrogenase and the GDSL-motif lipase hydrolase family protein. NADPH-dependent flavin reductase and beta-hydroxyacyl-ACP dehydratase were suppressed.

Creatine administration prevents Na +,K +-ATPase inhibition induced by intracerebroventricular administration of isovaleric acid in cerebral cortex of young rats

Isovaleric acidemia (IVAcidemia) is an inborn error of metabolism due to deficiency of isovaleryl-CoA dehydrogenase activity, leading to predominant accumulation of isovaleric acid (IVA). Patients affected by IVAcidemia suffer from acute episodes of encephalopathy, whose underlying mechanisms are poorly known. In the present study we investigated whether an intracerebroventricular injection of IVA could compromise energy metabolism in cerebral cortex of young rats. IVA administration significantly inhibited 14CO 2 production from acetate (22%) and citrate synthase activity (20%) in cerebral cortex homogenates prepared 24 h after injection. However, no alterations of these parameters were observed 2 h after injection. In contrast, no significant differences were found in the activities of succinate dehydrogenase, isocitrate dehydrogenase, electron transfer chain complexes or creatine kinase in rats sacrificed 2 or 24 h after IVA administration. Moreover, IVA injection significantly inhibited Na +,K +-ATPase activity (25%) in cerebral cortex of rats 2 or 24 h after IVA administration, while pre-treatment of rats with creatine completely prevented the inhibitory effects of IVA on Na +,K +-ATPase. In conclusion, in vivo administration of IVA inhibits the citric acid cycle probably through the enzyme citrate synthase, as well as Na +,K +-ATPase, a crucial enzyme responsible for maintaining the basal potential membrane necessary for a normal neurotransmission. It is presumed that inhibition of these activities may be involved in the pathophysiology of the neurological dysfunction of isovaleric academic patients. The present findings are of particular interest because treatment with creatine supplementation may represent a potential novel adjuvant therapeutic strategy in IVAcidemia.

Essential fatty acid profiling for routine nutritional assessment unmasks adrenoleukodystrophy in an infant with isovaleric acidaemia

We report a 16-month-old asymptomatic male with enzyme confirmed isovaleric acidaemia (IVA; isovaleryl-CoA dehydrogenase deficiency; OMIM 243500) who, upon routine nutritional follow-up, presented evidence of peroxisomal dysfunction. The newborn screen (2 days of life) revealed elevated C(5)-carnitine (2.95 μmol/L; cutoff <0.09 μmol/L) and IVA was subsequently confirmed by metabolic profiling and in vitro enzymology. Plasma essential fatty acid (EFA) analysis, assessed to evaluate nutritional status during protein restriction and L: -carnitine supplementation, revealed elevated C(26:0) (5.0 μmol/L; normal <1.3). Subsequently, metabolic profiling and molecular genetic analysis confirmed X-linked adrenoleukodystrophy (XALD). Identification of co-inherited XALD with IVA in this currently asymptomatic patient holds significant treatment ramifications for the proband prior to the onset of neurological sequelae, and critically important counselling implications for this family.

Acute decompensation of isovaleric acidemia induced by Graves’ disease

Sir, We present the first report of a metabolic exacerbation in a late onset form of isovaleric acidaemia (IVA, MIM 243500) due to hyperthyroidism. A 24-year-old woman was hospitalized for vomiting, drowsiness and a weight loss of 5 kg in 3 months. She had been treated since 7 years old for IVA with oral L-carnitine and had not experienced IVA decompensation since diagnosis. Despite an anabolic therapy [IV glucose (400 g/day) and lipids (40 g/ day)], a low protein diet and IV L-carnitine and PO L-glycine, her neurological state worsened in 24 h (Glasgow coma score of 11) and she was transferred to the ICU. The clinical examination showed tachypnea, paroxysmal atrial fibrillation and flapping tremor. Laboratory findings, at admission, revealed metabolic ketoacidosis: (pH 7.25, bicarbonates 9.8 mmol/l), normal glycaemia 5 mmol/l and hyperammonemia 100 lmol/l (controls \30 lmol/l). Additionally, her plasma amino acid profile found hypoalaninemia 122 lmol/l (controls 218–474 lmol/ l) and hyperleucinemia 157 lmol/l (controls 98–142 lmol/l). These metabolic abnormalities normalized within the first 24 h. However, despite biological improvement, she developed severe confusional syndrome and permanent atrial fibrillation. Hyperthyroidism was then diagnosed: TSH 0.005 mU/l (controls 0.3–4 mU/l), T4 43 pmol/l (controls 10–26 pmol/l), T3 11.7 pmol/l (controls 3–7 pmol/l). A high level of circulating antibodies to TSH-receptor led to the diagnosis of Graves’ disease. Clinical recovery was achieved after initiating treatment with carbimazole and propanolol in conjunction with Lcarnitine, L-glycine and a low protein diet. IVA is a rare autosomal recessive inborn error of metabolism due to isovaleryl CoA dehydrogenase deficiency (EC 1.3.99.10), an enzyme of the leucine catabolism pathway (Fig. 1) [1]. Its deficiency will cause the accumulation of intermediate metabolites (Fig. 1). These accumulated toxic substrates are responsible for the primary pathological effects seen in IVA [1]. In organic acidemias, an inadequate gluconeogenic response has been suggested to partly account in the pathophysiology of the hyperketotic hypoglycaemia linked to relative hypoalaninemia [2]. Moreover, the accumulation of acyl-CoAs can inhibit the urea cycle, resulting in hyperammonemia [3]. Patients with isovaleric acidemia must be treated during acute attack with glucose and lipids infusion without proteins for 48 h [1]. The administration of Lglycine and L-carnitine is designed to help the removal of isovalerate through excreting the non-toxic byproducts: isovalerylglycine and isovalerylcarnitine [4]. While the emergency treatment is administered, the factor triggering decompensation (infection, fasting, poor diet compliance and accidental high protein intake) which has induced the metabolic exacerbation must be treated [1]. As leucine constitutes about 10% of the amino acid content of the Leucine

Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy

The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, ( iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min −1 mg −1 in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid β-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

Proteomic analysis of stargazer mutant mouse neuronal proteins involved in absence seizure

The stargazer (stg) mutant mouse, having mutation in stargazin, the calcium channel gamma2 subunit, exhibited several neurological disorders including spontaneous absence seizure, cerebellar ataxia, and head tossing. To understand the molecular pathogenic mechanism of the absence seizure resulted from the loss of stargazin function, the thalamic proteomes between control mouse and stg mouse were compared. We identified 12 proteins expressed differentially (> 1.6-fold) by fluorescence two-dimensional difference gel electrophoresis and tandem mass spectrometry. Six of them are involved in basic metabolism including energy metabolism, three in stress response, two in axonal growth regulation, and one in the endoplasmic reticulum processing. All except mortalin showed decreased level of expression in stg mouse. Two stress-related proteins, mouse stress induced phosphoprotein 1 and peroxiredoxin 6 exhibited reduced levels of expression in stg mouse, while the level of another stress protein, mortalin was increased. Analysis of oxidative protein carbonylation in thalamic proteome of stg mouse showed higher level of carbonylated proteins in stg mouse than in control mouse. Interestingly, down-regulation of stress protein mouse stress induced phosphoprotein 1, metabolic enzyme isovaleryl-CoA dehydrogenase, and the two in neuronal axon growth, collapsin response mediator protein 2 and fascin homolog 1 coincides with the results of our previous study on gamma-butyrolactone-induced transient absence seizure. Our results suggest that the pathogenesis mechanism underlying absence seizure may involve the molecular events contributed by these proteins.

2-Methylbutyryl-coenzyme A dehydrogenase deficiency: Functional and molecular studies on a defect in isoleucine catabolism

2-Methylbutyryl-CoA dehydrogenase (MBD; coded by the ACADSB gene) catalyzes the step in isoleucine metabolism that corresponds to the isovaleryl-CoA dehydrogenase reaction in the degradation of leucine. Deficiencies of both enzymes may be detected by expanded neonatal screening with tandem-mass spectrometry due to elevated pentanoylcarnitine (C5 acylcarnitine) in blood, but little information is available on the clinical relevance of MBD deficiency. We biochemically and genetically characterize six individuals with MBD deficiency from four families of different ethnic backgrounds. None of the six individuals showed clinical symptoms attributable to MBD deficiency although the defect in isoleucine catabolism was demonstrated both in vivo and in vitro. Several mutations in the ACADSB gene were identified, including a novel one. MBD deficiency may be a harmless metabolic variant although significant impairment of valproic acid metabolism cannot be excluded and further study is required to assess the long-term outcome of individuals with this condition. The relatively high prevalence of ACADSB gene mutations in control subjects suggests that MBD deficiency may be more common than previously thought but is not detected because of its usually benign nature.

The Promoter Activity of Isovaleryl-CoA Dehydrogenase-Encoding Gene (ivdA) fromAspergillus oryzaeIs Strictly Repressed by Glutamic Acid

We cloned the isovaleryl-CoA dehydrogenase (IVD)-encoding gene from Aspergillus oryzae. The promoter of ivdA was subjected to beta-glucuronidase (GUS) reporter assays in which certain amino acids were used as a major carbon source. L-leucine most strongly induced GUS-activity, while in the case of L-glutamate, significantly low activity was found, indicating that ivdA transcription was strongly repressed by glutamic acid.

Different spectrum of mutations of isovaleryl-CoA dehydrogenase ( IVD) gene in Korean patients with isovaleric acidemia

Isovaleric acidemia (IVA) is an autosomal recessive inborn error of the leucine metabolism that is caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). Recent application of tandem mass spectrometry to newborn screening has allowed a significant expansion of the recognition of individuals with IVD deficiency. Although many patients have been reported worldwide, there are no genetically confirmed patients in Korea. This study characterizes IVD mutations in seven Korean IVA patients from six unrelated families. Bi-directional sequencing analysis identified two novel variations affecting consensus splice sites (c.144+1G>T in intron 1 and c.457-3_2CA>GG in intron 4) and three novel variations altering coding sequences (c.149G>T; Arg21Leu, c.832A>G; Ser249Gly, and c.1135T>G; Phe350Val). Five patients from four families were found to be compound heterozygotes while two unrelated patients were homozygous for the c.457-3_2CA>GG variation. Reverse-transcription polymerase chain reaction confirmed that both intron variations cause aberrant splicing. Furthermore, analysis of cultured lymphocyte extracts of the seven patients showed no detectable enzyme activity and reduced levels of IVD protein (<10.0% of control) in all samples. These results confirm IVD mutations in Korean patients with IVA and reveal that the mutation spectrum is different from previously reported patients.

Equine biochemical multiple acyl-CoA dehydrogenase deficiency (MADD) as a cause of rhabdomyolysis

Two horses (a 7-year-old Groninger warmblood gelding and a six-month-old Trakehner mare) with pathologically confirmed rhabdomyolysis were diagnosed as suffering from multiple acyl-CoA dehydrogenase deficiency (MADD). This disorder has not been recognised in animals before. Clinical signs of both horses were a stiff, insecure gait, myoglobinuria, and finally recumbency. Urine, plasma, and muscle tissues were investigated. Analysis of plasma showed hyperglycemia, lactic acidemia, increased activity of muscle enzymes (ASAT, LDH, CK), and impaired kidney function (increased urea and creatinine). The most remarkable findings of organic acids in urine of both horses were increased lactic acid, ethylmalonic acid (EMA), 2-methylsuccinic acid, butyrylglycine (iso)valerylglycine, and hexanoylglycine. EMA was also increased in plasma of both animals. Furthermore, the profile of acylcarnitines in plasma from both animals showed a substantial elevation of C4-, C5-, C6-, C8-, and C5-DC-carnitine. Concentrations of acylcarnitines in urine of both animals revealed increased excretions of C2-, C3-, C4-, C5-, C6-, C5-OH-, C8-, C10:1-, C10-, and C5-DC-carnitine. In addition, concentrations of free carnitine were also increased. Quantitative biochemical measurement of enzyme activities in muscle tissue showed deficiencies of short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and isovaleryl-CoA dehydrogenase (IVD) also indicating MADD. Histology revealed extensive rhabdomyolysis with microvesicular lipidosis predominantly in type 1 muscle fibers and mitochondrial damage. However, the ETF and ETF-QO activities were within normal limits indicating the metabolic disorder to be acquired rather than inherited. To our knowledge, these are the first cases of biochemical MADD reported in equine medicine.

Genetic mutation profile of isovaleric acidemia patients in Taiwan

Isovaleric acidemia (IVA), a rare recessive autosomal disorder, is caused by isovaleryl-CoA dehydrogenase (IVD) deficiency. IVA may present with symptoms during the acute stage of severe metabolic acidosis, ketosis, vomiting, and altered mental status. With the help of newborn screening (NBS) by tandem mass spectrometry (MS/MS), IVA can now be diagnosed presymptomatically. According to statistic data, the incidence of IVA in Taiwan was about 1/365,000. In this study, six IVA patients from five families were investigated and followed-up clinically. As for the timing, two patients were found before MS technique introduced to Taiwan, the others were identified after MS/MS applied to NBS. The blood level of C5-carnitine in our patients was 7.43–18.96 μM (with upper limit in our laboratory <0.51 μM) and all of their urines contained raised amounts of 3-hydrixyisovaleric acid and isovalerylglycine. Molecular analysis of their IVD gene revealed six mutation profiles, among which the 149G → A (Arg21His) and 1174 C → T (Arg363Cys) mutations have been reported previously, while the other four mutations, 386A → G (His100Arg), 347C → T (Ser87Phe), 1007G → A (Cys307Tyr) and 1199A → G (Tyr371Cys), were first reported. Specially, we found 1199A → G (Tyr371Cys) mutated was a common recurring missense mutation in our population (4 in 10 mutant alleles).

Proteome Mapping of the Effects of Age, Pressure Overload, and Aldosterone Antagonism on Cardiac Fibrosis

Aging, pressure overload, and aldosterone signaling have each been implicated as causes of cardiac fibrosis. Cardiac fibrosis, an excessive accumulation of extracellular matrix, promotes diastolic dysfunction and congestive heart failure. Treatment with spironolactone (SP), an aldosterone antagonist, is effective in attenuating cardiac fibrosis, but questions remain regarding the mechanisms of action and the efficacy of aldosterone antagonism in advanced age. PURPOSE: The purpose of the present study was to identify the independent and combined effects of age, aortic constriction, and aldosterone antagonism on the cardiac proteome. METHODS: Female F344 rats served as untreated controls (C) or were subjected to aortic constriction (AC) at 5 (Y) or 23 (O) months of age and treated with SP (30 mg/kg/day) or vehicle (V) for 6 months, yielding the following six groups: Y-C, Y-V, Y-SP, O-C, O-V, O-SP. Left ventricle free walls (LV) were harvested and quick-frozen in liquid nitrogen and were subsequently used to generate 2-dimensional electrophoretic (2-DE) proteome maps. PDQuest software was used to analyze SYPRO Orange-stained 2-DE gels, quantify spot densities, and compare expression levels between groups. Protein spots were identified with mass spectrometry of trypsin digests. Significance Analysis of Microarrays (SAM) was used to evaluate the protein expression levels, and a false discovery rate of <1% was used as a guideline to identify differentially expressed proteins. RESULTS: Proteins differentially expressed as a function of age included Immt-Er (−25%), dihydrolipoamide acetyltransferase (+173%), albumin (+40 to +64%), ATP synthase mitochondrial F0 complex (−39%), isovaleryl CoA dehydrogenase (−15%), acetyl-CoA dehydrogenase long-chain (−13%), creatine kinase (−20 to −33%), gluathione S-transferase (−44%), and ATP synthase mitochondrial F1 complex (+865%). Aortic constriction caused changes in lactate dehydrogenase B (+73%) and myoglobin (+73%). Spironolactone induced changes in myosin light polypeptide 3 (−53), albumin (−23 to −55%), isovaleryl CoA dehydrogenase (−34%), malate dehydrogenase (+42%), and creatine kinase (+30%). CONCLUSIONS: Aging and pressure overload are associated with a marked change in the stoichiometry of a wide range of metabolic proteins. Spironolactone treatment had mixed effects, exacerbating the age-associated decrease of some metabolic proteins, but increasing levels of other metabolic proteins. Supported by NIH R03 AG022625 and the Undergraduate Research Opportunity Program at the University of Michigan

Isovaleric acidemia: New aspects of genetic and phenotypic heterogeneity

Isovaleric acidemia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by a deficiency of the mitochondrial enzyme isovaleryl-CoA dehydrogenase (IVD) resulting in the accumulation of derivatives of isovaleryl-CoA. It was the first organic acidemia recognized in humans and can cause significant morbidity and mortality. Early diagnosis and treatment with a protein restricted diet and supplementation with carnitine and glycine are effective in promoting normal development in severely affected individuals. Both intra- and interfamilial variability have been recognized. Initially, two phenotypes with either an acute neonatal or a chronic intermittent presentation were described. More recently, a third group of individuals with mild biochemical abnormalities who can be asymptomatic have been identified through newborn screening of blood spots by tandem mass spectrometry. IVD is a flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron transfer flavoprotein. Human IVD has been purified from tissue and recombinant sources and its biochemical and physical properties have been extensively studied. Molecular analysis of the IVD gene from patients with IVA has allowed characterization of different types of mutations in this gene. One missense mutation, 932C>T (A282V), is particularly common in patients identified through newborn screening with mild metabolite elevations and who have remained asymptomatic to date. This mutation leads to a partially active enzyme with altered catalytic properties; however, its effects on clinical outcome and the necessity of therapy are still unknown. A better understanding of the heterogeneity of this disease and the relevance of genotype/phenotype correlations to clinical management of patients are among the challenges remaining in the study of this disorder in the coming years.

Functional analysis of acyl-CoA dehydrogenase catalytic residue mutants using surface plasmon resonance and circular dichroism

The acyl-CoA dehydrogenases (ACDs) are a family of flavoenzymes involved in the metabolism of fatty acids and branched-chain amino acids. The ACDs share a similar structure and a common dehydrogenation mechanism in which a catalytic glutamate extracts a proton from an acyl-CoA substrate. The resulting charge-transfer complex subsequently passes electrons to electron-transferring flavoprotein (ETF). We previously generated catalytic residue mutants of human short-chain acyl-CoA dehydrogenase (SCAD) and isovaleryl-CoA dehydrogenase (IVD) that were difficult to characterize by traditional methods. In the present study, we developed a novel surface plasmon resonance-based assay to measure substrate binding to these mutants. Replacement of the catalytic glutamate in either SCAD or IVD with glycine resulted in a several-fold reduction in affinity for substrate. Circular dichroism studies substantiated our earlier findings that both SCAD E368G and IVD E254G are unable to form a charge-transfer complex with substrate/product. The CD spectra of IVD E254G also indicated a perturbation of the flavin environment, a finding supported by molecular modeling that predicted a shift in the conformation of a conserved tryptophan that lies in close proximity to the flavin. Lastly, competitive inhibition studies using the ETF fluorescence reduction assay suggested that SCAD E368G and IVD E254G do not effectively compete with the wild-type enzymes for the physiological electron acceptor ETF.

Neonatal Screening by Tandem Mass Spectrometry

After completing this article, readers should be able to: 1. List the major categories of diseases detectable by expanded newborn screening. 2. Give examples of the benefits and limitations of screening by tandem mass spectrometry. 3. Describe the appropriate follow-up for an abnormal screening result. The screening of newborns for inherited metabolic disorders is a well-established public health activity, first implemented in the early 1960s for the presymptomatic identification of patients who have phenylketonuria (PKU). Since then, significant technological advances, most importantly the development of tandem mass spectrometry (MS/MS), have enabled the detection of an increasing number of metabolic disorders in newborn blood. This article reviews the basic technology of MS/MS and its application to newborn screening, with the aim of highlighting the benefits and pointing out some limitations of this powerful technology. Early screening for PKU used the bacterial inhibition assay of Guthrie, in which inhibition of bacterial growth on an agar medium, introduced by the addition of a phenylalanine analog, was overcome by the presence of dried blood discs containing high levels of phenylalanine (such as from a PKU patient). This approach later was adapted for the detection of leucine in patients who have maple syrup urine disease (MSUD), methionine in patients who have classic homocystinuria, and tyrosine in patients who have tyrosinemia. Over the years, other techniques have been implemented to screen for additional disorders, including congenital hypothyroidism, biotinidase deficiency, galactosemia, hemoglobinopathies, and congenital adrenal hyperplasia. The classic criteria used to determine whether a disorder is suitable for screening include: 1) the disorder is clinically and biochemically well defined, 2) it is associated with significant morbidity or mortality, 3) an effective treatment is available, and 4) there is a simple and safe screening test. (1) By the early 1990s, all states offered screening for at least PKU and congenital hypothyroidism, …

Stabilization of Non-productive Conformations Underpins Rapid Electron Transfer to Electron-transferring Flavoprotein

Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.

Identification of 19 New Metabolites Induced by Abnormal Amino Acid Conjugation in Isovaleric Acidemia

Isovaleric acidemia (IVA) is an autosomal recessive genetic disorder of the enzyme isovaleryl-CoA dehydrogenase, which is involved in leucine metabolism (1). Clinical symptoms include poor feeding, tachypnea, vomiting (2), listlessness, lethargy, coma (2)(3), and dehydration (4). Individuals homozygous for this defect are characterized primarily by the excretion of isovalerylglycine (2). The metabolic profile may be further complicated by intermediate metabolites such as 3- and 4-hydroxyisovaleric acid (5), methylsuccinic acid (6), methylfumaric acid (7), isovalerylglucuronide (8), isovalerylglutamic acid (9), N -isovalerylalanine and N -isovalerylsarcosine (10), isovalerylcarnitine (11), 3-hydroxyisoheptanioc acid (12), and alloisoleucine (4). Diagnosis of IVA is based on clinical symptoms and the presence of isovalerylglycine and 3-hydroxyisovaleric acid (13), with some of the above-mentioned metabolites also occurring to a greater or lesser extent (4). Despite the large number of already identified excreted metabolites, their occurrence still does not entirely explain all of the clinical symptoms experienced by these patients. The statement made in 1982 by Duran et al. (14) that “[t]he continuing search for ‘new’ metabolites may eventually lead to a better understanding of the relationship between the clinical conditions and their biochemical abnormalities” is the motivation for our search for previously unidentified metabolites in IVA. In this study, we analyzed the urine of IVA patients for the presence of induced amino acid metabolites not previously identified in IVA. Most of the amino acid conjugates occurring in the …

Convergent Evolution of a 2-Methylbutyryl-CoA Dehydrogenase from Isovaleryl-CoA Dehydrogenase in Solanum tuberosum

The potato cDNAs Solanum tuberosum isovaleryl-CoA dehydrogenases 1 and 2 (St-IVD1 and St-IVD2) encode proteins that are 84% identical to each other and 65 and 64% identical to human IVD, respectively. St-IVD2 protein was previously partially purified from potato tubers and confirmed to be an IVD. The function of St-IVD1 is unknown. In these experiments, both proteins were expressed in Escherichia coli and purified as intact homotetramers. The substrate preference profile of the St-IVD2 protein was similar to that of human IVD. However, recombinant St-IVD1 had maximal activity with 2-methylbutyryl-CoA, which in humans is dehydrogenated by short/branched-chain acyl-CoA dehydrogenase (SBCAD). Whereas molecular modeling predicts that the 2-methylbutyryl-CoA dehydrogenase (2MBCD) and IVD substrate binding pockets are nearly identical, 2MBCD has amino acid substitutions at five residues that are invariant among all of the known and putative IVDs. Site-directed mutagenesis was used to match the human IVD active site with that of potato 2MBCD. The resulting mutant IVD had detectable activity with 2-methylbutyryl-CoA and no activity with isovaleryl-CoA. The 2MBCD active site was compared with that of human SBCAD using molecular modeling. Residues Met-361 and Ala-365 of 2MBCD appear to partially substitute for the function of Tyr-380 in human SBCAD, binding the methyl branch linked to C2 of 2-methylbutyryl-CoA, whereas residues Val-88, Val-92, and Val-96 appear to bind the distal C4 methyl group. The presence of a 2MBCD in potato that is highly homologous to IVD is an example of convergent evolution within the acyl-CoA dehydrogenase family, leading to the independent occurrence of two enzymes (SBCAD and 2MBCD) specific for 2-methylbutyryl-CoA.