Functional Role of the Active Site Glutamate-368 in Rat Short Chain Acyl-CoA Dehydrogenase

Battaile, Kevin P.; Mohsen, Al‐Walid; Vockley, Jerry

doi:10.1021/bi961113r

Cited by 25 publications

(36 citation statements)

References 22 publications

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“…In the acyl-CoA dehydrogenases, the effect of the reverse mutation varies among the different enzymes. Replacing the glutamate with an aspartate in the rat short chain acyl-CoA dehydrogenase reduces the k cat /K m values for various substrates less than 3-fold, yet the same mutation in human isovaleryl-CoA dehydrogenase renders its activity toward its native substrate barely detectable (21). In the case of human liver medium chain acyl-CoA dehydrogenase, the E376D mutation decreases the rate of flavin reduction by 800-fold (22).…”

Section: Discussionmentioning

confidence: 99%

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,

Valley¹,

Fitzpatrick

2003

Biochemistry

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The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG ‡ for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.The flavoenzyme nitroalkane oxidase (NAO) 1 catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with consumption of oxygen and release of nitrite and hydrogen peroxide (Scheme 1) (1). While several other flavoprotein oxidases are able to oxidize nitroalkanes, they require the preformed anion as a substrate in a clearly nonphysiological reaction (2). NAO is unique in that it uses the neutral forms of nitroalkanes as substrates (3); moreover, the enzyme is induced by growth of Fusarium oxysporum on nitroethane, consistent with nitroalkane oxidation being the physiological role of the enzyme (1). NAO can oxidize a broad range of primary and secondary nitroalkanes, although primary aliphatic nitroalkanes are the best substrates (4).As is the case with many flavoproteins, the reaction of NAO can be divided into reductive and oxidative half-reactions (5). In the reductive half-reaction, the nitroalkane substrate reacts with the oxidized flavin to generate the oxidized product and reduced flavin. The mechanism of this half-reaction (Scheme 2) has been proposed to involve removal of the substrate α-hydrogen as a proton, followed by attack of the resulting carbanion on the flavin (6). Loss of nitrite from the resulting adduct would generate an electrophilic species to which hydroxide adds (path a); this flavin adduct then decomposes to form reduced flavin and aldehyde or ketone. Evidence for this mechanism comes from the demonstration that an inactive 5-nitrobutyl-FAD adduct

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Section: Discussionmentioning

confidence: 99%

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,

Valley¹,

Fitzpatrick

2003

Biochemistry

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“…Hypothetical reading frames found in sequences retrieved from several strains of E. coli (e.g. accession ZP_00726504 from strain F11) possess a specific glutamine residue and context within the C-terminal domain, known to be important in the active site of enoyl-CoA dehydrogenases (Becker et al 1993;Djordjevic et al 1995;Thorpe et al 1995;Battaile et al 1996) (M.D. Redwood, unpublished work).…”

Section: Enterobacterial Phb and Implications For Phb Manufacturementioning

confidence: 99%

Polyhydroxybutyrate accumulation by a Serratia sp.

et al. 2007

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A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbonlimitation in continuous culture. 1.3 mmoles of citrate were consumed per 450 mg of biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry weight. The inclusion bodies were stained with Sudan Black and Nile Red, suggesting a lipid material which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by gas chromatography and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with Nile Red fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increased overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.

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“…Anaerobic Titration of Wild Type and Mutant SBCADs with Substrate-Wild type and mutant enzymes were titrated with (S)-2-methylbutyryl-CoA or hexanoyl-CoA as described previously (20,30,35) with minor modifications. Spectral scans were performed using a Beckman DU7400 spectrophotometer (Palo Alto, CA) equipped with a computer.…”

Section: Construction Of Sbcad Prokaryotic Expression Vectors-mentioning

confidence: 99%

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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Functional Role of the Active Site Glutamate-368 in Rat Short Chain Acyl-CoA Dehydrogenase

Cited by 25 publications

References 22 publications

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,

Polyhydroxybutyrate accumulation by a Serratia sp.

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

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Functional Role of the Active Site Glutamate-368 in Rat Short Chain Acyl-CoA Dehydrogenase

Cited by 25 publications

References 22 publications

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate,

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate,

Polyhydroxybutyrate accumulation by a Serratia sp.

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

Contact Info

Product

Resources

About

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,