Identification of the catalytic residue of human short/branched chain acyl-CoA dehydrogenase by in vitro mutagenesis

Binzak, Barbara A.; Willard, Jan; Vockley, Jerry

doi:10.1016/s0167-4838(97)00161-1

Cited by 17 publications

(17 citation statements)

References 24 publications

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“…Reduction of the ϳ450-nm maximum with (S)-2-methylbutyryl-CoA was similar for both wild type enzymes at a 1:1 substrate ratio, and neither showed complete reduction even with saturating concentrations. This is in agreement with our findings reported previously (22). Addition of hexanoyl-CoA to human SBCAD at a 1:1 molar ratio, in contrast, led to significantly greater reduction of this peak than was seen with the rat enzyme (5 versus 44%, respectively), although saturating substrate concentrations caused equal reduction of both enzymes.…”

Section: Kinetic Properties Of Purified Wild Type and Mutantsupporting

confidence: 93%

“…Isobutyryl-CoA and hexanoylCoA were purchased from Sigma. (S)-2-Methylbutyryl-CoA was synthesized as described previously (22). The protein concentration in the reaction mixture was adjusted to give a linear reaction.…”

Section: Construction Of Sbcad Prokaryotic Expression Vectors-mentioning

confidence: 99%

“…Previous studies have shown that the position of the active site catalytic glutamate, located at two different amino acid positions in the primary carbon backbone in the various ACDs, affects substrate specificity (20 -22). These residues, however, are in the same position in rat and human SBCAD indicating that other amino acid residues contribute to the substrate specificity of SBCAD (22). Therefore, we have begun using SBCAD as a model to study the amino acid residues and motifs important in determining acyl-CoA substrate specificity.…”

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confidence: 99%

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A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Burghardt

Vockley

2003

Journal of Biological Chemistry

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Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to ␤-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.The acyl-CoA dehydrogenases (ACDs) 1 are a family of related enzymes that catalyze the ␣, ␤-dehydrogenation of acylCoA esters, transferring electrons to electron transferring flavoprotein (1-4). Deficiencies of these enzymes are important causes of human disease (3-7). Biochemical and immunological studies have identified at least 9 distinct members of this enzyme family, each having a characteristic substrate utilization pattern (8 -13). Very long, long, medium, and short chain acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively) catalyze the first step in the mitochondrial ␤-oxidation of fatty acids with substrate optima of 16, 16, 8, and 4 carbon chains, respectively (3, 14 -15). A new ACD has been identified recently (ACAD9) that is also active against long chain acyl-CoA substrates (16). Isovaleryl-CoA dehydrogenase (IVD), short/branched chain acyl-CoA dehydrogenase (SBCAD, also known as 2-methyl branched chain acyl-CoA dehydrogenase), and isobutyryl-CoA dehydrogenase (IBD) catalyze the third step in leucine, isoleucine, and valine metabolism, respectively (2-4, 11, 12, 17, 18), whereas glutaryl-CoA dehydrogenase is active in lysine metabolism (19).We have shown previously that SCAD, SBCAD, and IBD can all utilize butyryl-CoA as substrate (albeit with different efficiencies), whereas ...

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Section: Kinetic Properties Of Purified Wild Type and Mutantsupporting

confidence: 93%

Section: Construction Of Sbcad Prokaryotic Expression Vectors-mentioning

confidence: 99%

mentioning

confidence: 99%

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A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Burghardt

Vockley

2003

Journal of Biological Chemistry

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“…Glycine residues at precursor positions 116, 186, 211, 243, 266, 297, 318, 406, 431 (75, 144, 165, 204, 215, 245, 264, 352, 355 in the mature IBDH sequence) are highly conserved in all of the branched chain ACDs. The position of the presumed catalytic base of the enzymes is also highly conserved (numbered as residue in mature protein): Glu381 for human SBCADH [11], Glu368 for human SCADH [17], and Glu254 for human IVDH [18,23]. The divergence of the position of the catalytic base in IVDH suggests that it is evolutionarily more distant from the other family members.…”

Section: Discussionmentioning

confidence: 99%

“…More recently, Molecular Genetics and Metabolism 77 (2002) 68-79 www.academicpress.com the rat and human cDNAs for this enzyme have been cloned and the gene was named short-branched chain acyl-CoA dehydrogenase (ACADSB; see Table 1 for a summary of genetic nomenclature and protein designations) [6,7,11]. Recombinant rat SBCADH produced in Escherichia coli, like its native counterpart, could efficiently utilize both isobutyryl-and 2-methylbutyryl-CoA as substrate.…”

Section: Introductionmentioning

confidence: 99%

Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans

Nguyen

Andresen

Corydon

et al. 2002

Molecular Genetics and Metabolism

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The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the a,b-dehydrogenation of acyl-CoA esters. Two homologues active in branched chain amino acid metabolism have previously been identified. We have used expression in Escherichia coli to produce a previously uncharacterized ACD-like sequence (ACAD8) and define its substrate specificity. Purified recombinant enzyme had a k cat =K m of 0.8, 0.23, and 0.04 (lM À1 s À1 ) with isobutyryl-CoA, (S) 2-methylbutyryl-CoA, and n-propionylCoA, respectively, as substrates. Thus, this enzyme is an isobutyryl-CoA dehydrogenase. A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine. Amplified ACAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905G > A) in the ACAD8 coding region compared to the sequence from control cells. This encodes an Arg302Gln substitution in the full-length protein (position 280 in the mature protein), a position predicted by molecular modeling to be important in subunit interactions. The mutant enzyme was stable but inactive when expressed in E. coli. It was also stable and appropriately targeted to mitochondria, but inactive when expressed in mammalian cells. These data confirm further the presence of a separated ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase, IBDH), along with the first enzymatic and molecular confirmation of a deficiency of this enzyme in a patient. Ó

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Acyl‐CoADehydrogenases

Bartlett¹

2002

Wiley Encyclopedia of Molecular Medicine

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Identification of the catalytic residue of human short/branched chain acyl-CoA dehydrogenase by in vitro mutagenesis

Cited by 17 publications

References 24 publications

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans

Acyl‐CoADehydrogenases

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