Search

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. [ABSTRACT FROM AUTHOR]

3‐Hydroxyisobutyric acid (3HiB) is an intermediate in the degradation of the branched‐chain amino acid valine. Disorders in valine degradation can lead to 3HiB accumulation and its excretion in the urine. This article describes the first two patients with a new metabolic disorder, 3‐hydroxyisobutyrate dehydrogenase (HIBADH) deficiency, its phenotype and its treatment with a low‐valine diet. The detected mutation in the HIBADH gene leads to nonsense‐mediated mRNA decay of the mutant allele and to a complete loss‐of‐function of the enzyme. Under strict adherence to a low‐valine diet a rapid decrease of 3HiB excretion in the urine was observed. Due to limited patient numbers and intrafamilial differences in phenotype with one affected and one unaffected individual, the clinical phenotype of HIBADH deficiency needs further evaluation. [ABSTRACT FROM AUTHOR]

Enzymes related to β-hydroxyacid dehydrogenases/3-hydroxyisobutyrate dehydrogenases are ubiquitous, but most of them have not been characterized. An uncharacterized protein with moderate sequence similarities to Gluconobacter oxydans succinic semialdehyde reductase and plant glyoxylate reductases/succinic semialdehyde reductases was found in the genome of Acetobacter aceti JCM20276. The corresponding gene was cloned and expressed in Escherichia coli. The gene product was purified and identified as a glyoxylate reductase that exclusively catalyzed the NAD(P)H-dependent reduction of glyoxylate to glycolate. The strict substrate specificity of this enzyme to glyoxylate, the diverged sequence motifs for its binding sites with cofactors and substrates, and its phylogenetic relationship to homologous enzymes suggested that this enzyme represents a novel class of enzymes in the β-hydroxyacid dehydrogenase family. This study may provide an important clue to clarify the metabolism of glyoxylate in bacteria. Abbreviations: GR: glyoxylate reductase; GRHPR: glyoxylate reductase/hydroxypyruvate reductase; HIBADH: 3-hydroxyisobutyrate dehydrogenase; SSA: succinic semialdehyde; SSAR: succinic semialdehyde reductase AceGR is NAD(P)H-dependent glyoxylate reductase representing a novel class of enzymes in the β-hydroxyacid dehydrogenase family. [ABSTRACT FROM AUTHOR]

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) family catalyzes the NAD + - or NADP + -dependent oxidation of various β-hydroxyacid substrates into their cognate semialdehydes for diverse metabolic pathways. Because HIBADH group members exhibit different substrate specificities, the substrate-recognition mode of each enzyme should be individually characterized. In the current study, we report the biochemical and structural analysis of a HIBADH group enzyme from Bacillus cereus (bcHIBADH). bcHIBADH mediates a dehydrogenation reaction on S-3-hydroxyisobutyrate substrate with high catalytic efficiency in an NAD + -dependent manner; it also oxidizes l -serine and 3-hydroxypropionate with lower activity. bcHIBADH consists of two domains and is further assembled into a functional dimer rather than a tetramer that has been commonly observed in other prokaryotic HIBADH group members. In the bcHIBADH structure, the interdomain cleft forms a putative active site and simultaneously accommodates both an NAD + cofactor and a substrate mimic. Our structure-based comparative analysis highlights structural motifs that are important in the cofactor and substrate recognition of the HIBADH group. [ABSTRACT FROM AUTHOR]

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant ( k/ K) of 604.1 ± 71.1 mM/S on ( S)-3-hydroxyisobutyrate, but no detectable activity on ( R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD over NADP as a cofactor for its catalytic activity. The k/ K determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD at 37°C and pH 9.0. In addition to ( S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl- d, l-serine, and methyl-( S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k/ K values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn increased the activity of 3HIBDHI significantly, whereas the presence of Fe, Hg and Ag in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation. [ABSTRACT FROM AUTHOR]

We have cloned a gene encoding an aldehyde oxidase (ALOD) oxidized glyoxal but not glyoxylic acid from Pseudomonas sp. AIU 362. The ALOD gene contained an open reading frame consisting of 888 nucleotides corresponding to 295 amino acid residues. The deduced amino acid sequence exhibited a high similarity to those of 3-hydroxyisobutyrate dehydrogenases (3-HIBDHs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant ALOD expressed in E. coli BL21 was 20,000-fold higher than that of ALOD in Pseudomonas sp. AIU 362. The recombinant ALOD exhibited ALOD activity and 3-HIBDH activity. The 3-HIBDH from Pseudomonas putida KT2440 also exhibited ALOD activity. Thus, the ALOD from Pseudomonas sp. AIU 362 and 3-HIBDH from P. putida KT2440 were classified into the same enzyme group. [ABSTRACT FROM AUTHOR]

The branched-chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy. [ABSTRACT FROM AUTHOR]

Kinetic analysis of substrate specificity revealed that a recombinant Arabidopsis protein catalyzes the conversion of glyoxylate to glycolate (Km,glyoxylate = 4.5 μmol·L–1) and succinic semialdehyde (SSA) to γ-hydroxybutyrate (Km, SSA = 0.87 mmol·L–1) via an essentially irreversible, NADPH-based mechanism. In this report, the enzyme was further characterized via initial-velocity, dead-end inhibition and product inhibition studies. The kinetic mechanism was ordered Bi Bi, involving the complexation of NADPH to the enzyme before glyoxylate or SSA, and the release of NADP+ before glycolate or γ-hydroxybutyrate, respectively. It can be concluded that the enzyme functions as a NADPH-dependent glyoxylate reductase (EC 1.1.1.79) or possibly an aldehyde reductase (EC 1.1.1.2), and the kinetic mechanism involved is consistent with that found in members of both the aldo-keto reductase and 3-hydroxyisobutyrate dehydrogenase-related superfamilies of enzymes. Since NADP+ was an effective competitive inhibitor with respect to NADPH (Ki = 1–3 µmol·L–1), it is proposed that the ratio of NADPH/NADP+ regulates enzymatic activity in planta. [ABSTRACT FROM AUTHOR]

Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.Cell Death and Differentiation (2007) 14, 348–357. doi:10.1038/sj.cdd.4402001; published online 23 June 2006 [ABSTRACT FROM AUTHOR]

Abstract: 3-Hydroxyisobutyric aciduria is a rare entity and affected individuals display a range of clinical manifestations including dysmorphic features and neurodevelopmental problems in the majority of patients. Here, we present two novel patients with 3-hydroxyisobutyric aciduria. To our knowledge, these are the 11th and 12th cases of 3-hydroxyisobutyic aciduria reported. It is believed that a deficiency in 3-hydroxyisobutyrate dehydrogenase is the most likely cause of this disorder. Measurement of 3-hydroxyisobutyrate dehydrogenase activity in fibroblasts homogenates of the two newly identified patients and a previously reported patient, however, revealed similar activities as in control fibroblasts. Since other enzymes with overlapping substrate specificity could conceal abnormal 3-hydroxyisobutyrate dehydrogenase activity, we cloned a candidate human cDNA for 3-hydroxyisobutyrate dehydrogenase (HIBADH). By heterologous expression in Escherichia coli, we showed that the product of the HIBADH gene indeed displays 3-hydroxyisobutyrate dehydrogenase activity. Mutation analysis of the corresponding gene in the patients suffering from 3-hydroxyisobutyric aciduria revealed no mutations. We conclude that HIBADH is not the causative gene in 3-hydroxyisobutyric aciduria. [Copyright &y& Elsevier]

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme. [Copyright &y& Elsevier]

NADPH-dependent glyoxylate reductases from Arabidopsis thaliana (AtGLYR) convert both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. The primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the β-HAD (β-hydroxyacid dehydrogenase) protein family, whose members include 3-hydroxyisobutyrate dehydrogenase, tartronate semialdehyde reductase and 6-phosphogluconate dehydrogenase. Here, site-directed mutagenesis was utilized to identify catalytically important amino acid residues for glyoxylate reduction in AtGLYR1. Kinetic studies and binding assays established that Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis. The low activity of the mutant enzymes precluded kinetic studies with succinic semialdehyde. The crystal structure of AtGLYR1 in the absence of substrate was solved to 2.1Å by molecular replacement using a previously unrecognized member of the β-HAD family, cytokine-like nuclear factor, thereby enabling the 3-D structure of the protein to be modeled with substrate and co-factor. Structural alignment of AtGLYR1 with β-HAD family members provided support for the essentiality of Lys170, Phe173, Asp239, Ser121, Asn174 and Thr95 in the active site and preliminary support for an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule. This information established that AtGLYR1 is a member of the β-HAD protein family. Sequence and activity comparisons indicated that AtGLYR1 and the plastidial AtGLYR2 possess structural features that are absent in Arabidopsis hydroxypyruvate reductases and probably account for their stronger preference for glyoxylate over hydroxypyruvate., (© 2013.)