Dehydrogenation Of L And D3HydroxyacylcoA

The dehydrogenation of L- and D-3-hydroxyacyl-CoAs is catalyzed by L- and D-3-hydroxyacyl-CoA dehydrogenases, respectively. These enzymes display strict substrate stereochemical specificity, but they both produce 3-ketoacyl-CoAs. The L-3-hydroxyacyl-CoA dehydrogenase binds its coenzyme NAD+ and its substrate to a cleft between its N- and C-terminal domains.23'24 The crystal structure of pig heart L-3-hydroxyacyl-CoA dehydrogenase was published a decade ago,23 but the catalytic mechanism of this type of dehydrogenase was not known until we identified a conserved histidine as the catalytic residue. L-3-Hydroxyacyl-CoA dehydrogenases are associated with the C-terminal region of multifunctional proteins except for those in the mitochondrial matrix6'7'26 (see Table 1). In contrast, D-3-hydroxyacyl-CoA dehydrogenases are associated with the N-terminal region of peroxisomal multifunctional proteins.3'8'9

The replacement of glycine-322 with alanine in the large subunit of the E. coli fatty acid oxidation complex significantly increased the Km value for NADH, so the importance of a glycine-rich sequence (GXGXXG) to the coenzyme binding was confirmed in a catalytic context.25 Since the pH dependence of the kcJKm of the E. coli dehydrogenase suggested the catalytic involvement of an amino acid residue with a neutral pKa, we replaced histidine-450' which is the only histidine conserved in all known L-3-hydroxyacyl-CoA dehydrogenases, with either glutamine or alanine by site-directed mutagenesis. Both mutant dehydrogenases showed a more than 1,500-fold decrease in the kca„ but their Km values and the catalytic properties of other component enzymes of the fatty acid oxidation complex exhibited only a very mild change. As a result, the imidazole of histidine-450, which is located in a loop between p-strands 6 and 7, was found to be the functional group.25 Thereafter' a conserved glutamate-462 was identified by "alanine-scanning" mutagenesis as another important element involved in dehydrogenase catalysis. The electrostatic interaction between this acidic residue and the catalytic residue forms a catalytic His450-Glu462 pair (Figure 3). Such His-Glu couples play a pivotal role in the catalytic process of L-3-hydroxyacyl-CoA dehydrogenases by maintaining the electroneutrality in the active site and reducing the activation energy of the reaction.27 The amino acid sequences of all known L-3-hydroxy acyl-CoA dehydrogenases are highly conserved in this key region of the active center' and the consensus sequence has been designated as the signature pattern of the L-3-hydroxyacyl-CoA dehydrogenase family6 (Fig. 4). The catalytic mechanism delineated in Fig. 3 is applicable to other members of the dehydrogenase family, including long-chain L-3-hydroxyacyl-CoA dehydrogenase of the mitochondrial inner membrane-bound trifunctional p-oxidation complex, which is more closely related evolutionarily to the E. coli fatty acid oxidation complex than to the corresponding matrix p-oxidation enzymes, in spite of a functional cooperation of the two mitochondrial P-oxidation systems in the same organelle.28

The negative charge of glutamate-462 was found to be necessary for increasing the thermostability of the multienzyme complex, and amidation of the y-carboxyl group of glutamate-462 is known to have an adverse effect on the 3-ketoacyl-CoA thiolase activity associated with the small subunit of the fatty acid oxidation complex.27 These findings provided evidence that a Glu510 Gin mutation of mitochondrial trifunctional P-oxidation complex, which corresponds to the Glu462 —> Gin mutation described above,

Table 1. Different fatty acid ß-oxidation enzyme systems"

Reaction

Bacteria

Mitochondrial inner membrane

Mitochondrial matrix

Animal Plant peroxisome glyoxysome

Fungi

(S. cerevisial)

1. Dehydrogenation acyl-CoA

dehydrogenase very long-chain acyl-CoA

dehydrogenase various acyl-CoA

dehydrogenase11

acyl-CoA oxidase acyl-CoA oxidase acyl-CoA oxidase

2. Hydration

3. Dehydrogenation long-chain enoyl-CoA hydratasec fatty acid oxidation <a¡2¡Oí_^

m fiai large subunit multifunctional fatty acid oxidation Protein

4. Thiolytic cleavage mall subunit

3-ketoacyl-CoA thiolase

¡afunctional enoyl-CoA

ß-oxidation complex hydratase ¡urge subunit \

long chain enoyl-CoA hydratase :3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

/âmall subunit long chain 3-ketoacyl-CoA thiolase

L-3-hydroxyacyl-CoA

dehydrogenase4

short chain L-3-

hydroxyacyl-CoA

dehydrogenase'

3-ketoacyl-CoA thiolase*

trifunctional

ß-oxidation enorme'

3-ketoacyl-

thiolase

N r tetrafunctional ß-oxidation enzyme

D-specific bifunctional ß-oxidation enzyme

3-ketoacyl-CoA thiolase

3-ketoatyl-CoA thiolase a. Some bacteria and fungi have unusual B-oxidation systems different from those presented here.

b. The existence of branched-diain acyl-CoA dehydrogenases as well as medium-chain acyl-CoA dehydrogenase and its "isoenzymes" differing in their preferences to the chain-lengths of the substrates brings additional complexity to the B-oxidation system.1

c. This enzyme has not yet been purified d. The sequence difference between pig liver and heart dehydrogenases suggest that there may be tissue-specific isoenzymes.6

e. This enzyme is a new member of the short chain dehydrogenase family. It can catalyze the dehydrogenation of 1^3-hydroxyaql-CoAj with different chain-lengths.7

i Another multifunctional protein 2 is identical to 17 B-hydraxysteroid dehydrogenase type IV, which is presumably involved in steroid metabolism even though it displays D-3-hydroxyacyl-CoA dehydratase and dehydrogenase activities.** g. Aoetoaeetyi-CoA thiolase serves mainly for the metabolism of ketobodies.

Figure 3. Schematic diagram of a model of the active site of E. coli L-3-hydroxyacyl-CoA dehydrogenase. Glu462 is not directly involved in the binding of substrate or coenzyme. The negatively charged y-carboxyl group of Glu462 significantly reduces the free energy of the transition state due to an electrostatic interaction with the catalytic residue, His450, in the ternary complex, thus maintaining electroneutrality in the desolvated active center during catalysis. In the dehydrogenase • coenzyme • substrate ternary complex, His450 serves as a general catalytic base/acid to catalyze the carbonyl/alcohol interconversion of the substrates and the coupled redox reaction of NAD7NADH. The substrate takes such an orientation that a large part of the CoA moiety extends into the solution, and the nicotinamide ring of the coenzyme has "B-side" specificity.

Figure 3. Schematic diagram of a model of the active site of E. coli L-3-hydroxyacyl-CoA dehydrogenase. Glu462 is not directly involved in the binding of substrate or coenzyme. The negatively charged y-carboxyl group of Glu462 significantly reduces the free energy of the transition state due to an electrostatic interaction with the catalytic residue, His450, in the ternary complex, thus maintaining electroneutrality in the desolvated active center during catalysis. In the dehydrogenase • coenzyme • substrate ternary complex, His450 serves as a general catalytic base/acid to catalyze the carbonyl/alcohol interconversion of the substrates and the coupled redox reaction of NAD7NADH. The substrate takes such an orientation that a large part of the CoA moiety extends into the solution, and the nicotinamide ring of the coenzyme has "B-side" specificity.

is responsible for loss of long-chain dehydrogenase activity29 and rapid degradation of the |3-oxidation complex.30 The discovery of a catalytic His-Glu pair has contributed enormously to the understanding of the pathogenesis of long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency at the molecular level. In addition, patients with so-called isolated LCHAD deficiency29 were found to be deficient in long-chain 3-ketoacyl-CoA thiolase activity as well, and this was attributed to the same mutation at the large subunit.27

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    How to distinguish d and l hydroxyacyl coa?
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