Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity*

Michelle Treber, Jia Dai, and Gebre WoldegiorgisDagger

From the Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921

Received for publication, October 15, 2002, and in revised form, January 15, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine. To determine the role of the conserved glutamate residue, Glu-603, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in malonyl-CoA sensitivity, suggesting that these two conserved residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or the putative low affinity acyl-CoA/malonyl-CoA binding site.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Carnitine palmitoyltransferase I (CPTI)1 catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine, the first step in the transport of long chain fatty acids from the cytoplasm to the mitochondrial matrix, a rate-limiting step in beta -oxidation (1, 2). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a muscle isoform (M-CPTI), that are 62% identical in amino acid sequence (3-8). As an enzyme that catalyzes the first rate-limiting step in beta -oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA (1, 2), the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis. Because of its central role in fatty acid metabolism, understanding the molecular mechanism of the regulation of the CPT system is an important first step in the development of treatments for diseases, such as myocardial ischemia and diabetes, and in human inherited CPTI deficiency diseases (9-11).

We developed a novel high level expression system for human heart M-CPTI, rat L-CPTI, and CPTII in the yeast Pichia pastoris, an organism devoid of endogenous CPT activity (6, 12-14). Furthermore, by using this system, we have shown that CPTI and CPTII are active distinct enzymes and that L-CPTI and M-CPTI are distinct malonyl-CoA-sensitive CPTs that are reversibly inactivated by detergents. Recent site-directed mutagenesis studies from our laboratory have demonstrated that glutamic acid 3 and histidine 5 in L-CPTI are necessary for malonyl-CoA inhibition and high affinity binding but not for catalysis (15, 16). For M-CPTI, our mutagenesis studies demonstrate that in addition to Glu-3 and His-5, Val-19, Leu-23, and Ser-24 are necessary for malonyl-CoA inhibition and high affinity binding, in agreement with the differences in malonyl-CoA sensitivity observed between M-CPTI and L-CPTI (17). In addition, our site-directed mutagenesis studies of conserved residues in the C-terminal domain of L-CPTI demonstrated that conserved arginine and tryptophan residues are important for catalysis (18). In this report, our mutagenesis studies demonstrate for the first time that the conserved residues Arg-601, Glu-603, and Arg-606 in L-CPTI are important for catalytic activity and malonyl-CoA sensitivity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

L-CPTI mutants were constructed by the overlap extension PCR procedure using the primers shown in Table I with the wild type plasmid DNA (pGAP-L-CPTI) as template (12, 19). For example, to construct the E603Q mutant, the primers f-GWW3·r-E603Q and r-MDR2·f-E603Q were used to generate 900 bp and 600 bp PCR products, respectively, using the wild type L-CPTI cDNA as a template. The two PCR products were purified, mixed, and used as a template for a second-round PCR with the primers f-GWW3·r-MDR2. The 1.5-kb PCR product was digested with AvaI-SacI, and the DNA fragment was subcloned into AvaI-SacI-cut wild type L-CPTI cDNA in the pGAP expression vector. Bacterial colonies obtained upon transformation of the mutagenesis reactions were screened by PCR using the primer pair f-GWW3-r-E603QCK for Gln and f-GWW3-r-E603HCK for His and Asp. The R601A and R606A mutants were constructed as described previously (18). The mutations were confirmed by DNA sequencing.


                              
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Table I
PCR primers used for construction of L-CPTI mutants
E603HCK is also used to check the introduction of E603D mutation.

The expression plasmids were linearized by digestion with the restriction enzyme BspEI and integrated into the His4 locus of P. pastoris GS115 by electroporation (16). Histidine prototrophic transformants were selected on YND (yeast nitrogen base with dextrose) plates and grown on YND medium. Mitochondria were isolated by disrupting the yeast cells with glass beads (12) and used to monitor activity and malonyl-CoA sensitivity.

CPT Assay-- CPT activity was assayed by the forward exchange method using L-[methyl-3H]carnitine (12, 20). The Km value for palmitoyl-CoA was determined by varying the palmitoyl-CoA concentration from 2.8 to 225 µM at a fixed molar ratio (6.1:1) of palmitoyl-CoA to albumin as described previously (16). The concentration of carnitine was fixed at 200 µM. The Km for carnitine was determined by varying the carnitine concentration from 11 to 472 µM at a fixed 111 µM palmitoyl-CoA.

Western Blot-- Proteins were separated by SDS-PAGE in a 10% gel and transferred onto nitrocellulose membranes. Immunoblots were developed by incubation with the L-CPTI-specific antibodies as described previously (16).

Sources of other materials and procedures were as described in our previous publication (16).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preincubation of isolated mitochondria from the yeast strain expressing rat liver L-CPTI at room temperature with dicyclohexylcarbodiimide (DCCD), a glutamate-specific modifying reagent (21), resulted in an irreversible 50% loss in catalytic activity (data not shown). These preliminary chemical modification studies with DCCD provided evidence that a conserved glutamate residue(s) is important for maximal L-CPTI activity.

Sequence alignment of the sequences of all carnitine and choline acyltransferases from different species showed the presence of two conserved glutamate (E) residues (Fig. 1), Glu-590 and Glu-603. The conserved Glu-603 residue is flanked by two highly conserved arginine residues, Arg-601 and Arg-606, that we previously demonstrated were important for L-CPTI activity and malonyl-CoA sensitivity (18).


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Fig. 1.   Sequence alignment of portions of the C-terminal region of various acyltransferases. *, identical residues; :, conserved residues.

Generation of Mutations and Expression in P. pastoris-- Construction of plasmids carrying substitution mutations E590A, E603A, E603H, E603Q, and E603D was performed as described under "Experimental Procedures," and for R601A and R606A as described previously (18). P. pastoris was chosen as an expression system for L-CPTI and the mutants, because it does not have endogenous CPT activity (6, 12-16). The P. pastoris expression plasmids expressed L-CPTI under control of the P. pastoris glyceraldehyde-3-phosphate dehydrogenase gene promoter (12, 22). Yeast transformants with the wild type L-CPTI gene and the mutants were grown in liquid medium supplemented with glucose (12).

Western blot analysis of wild type L-CPTI (88 kDa) and the mutants using a polyclonal antibody directed against a maltose-binding protein·L-CPTI fusion protein (12) is shown in Fig. 2. For the wild type and the mutants E603A, E603Q, E603H, and E603D, proteins of predicted sizes were synthesized with similar steady-state levels of expression. Mutants R601A and R606A were also expressed at similar steady-state levels as reported previously (12).


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Fig. 2.   Immunoblot showing expression of wild type (lane 1), control without insert (lane 2), E603A (lane 3), E603Q (lane 4), E603H (lane 5), and E603D (lane 6) mutants in P. pastoris. Mitochondria (23-48 µg) from the yeast strains expressing the wild type and each of the point mutants were separated on a 10% SDS-PAGE and blotted onto a nitrocellulose membrane. The immunoblot was developed using L-CPTI-specific antibodies as described previously (16).

Effect of Mutations on L-CPTI Activity and Malonyl-CoA Sensitivity-- Substitution mutants E603A and E603H were inactive. A change of Glu-603 to Asp resulted in only a 33% loss in L-CPTI activity, but the mutant E603D exhibited a 15-fold decrease in malonyl-CoA sensitivity as shown by the IC50 values in Table II. Substitution of Glu-603 with glutamine resulted in a 92% loss in L-CPTI activity and a 14-fold decrease in malonyl-CoA sensitivity (Table II). We previously reported that mutation of the highly conserved arginine residues Arg-601 and Arg-606 to alanine resulted in >98 and 93% loss in L-CPTI activity, respectively (18). The loss in activity observed with the R601A and R606A mutants was also accompanied by decreased sensitivity to malonyl-CoA inhibition (18). Furthermore, our site-directed mutagenesis studies demonstrate that mutants R601A and R606A have an over 40-fold decrease in malonyl-CoA sensitivity compared with the wild type L-CPTI enzyme (Table II). In short, our studies identify for the first time three conserved residues in the C-terminal region of L-CPTI, Arg-601, Glu-603, and Arg-606, that are important for both catalytic activity and malonyl-CoA sensitivity.


                              
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Table II
CPT activity and malonyl-CoA sensitivity in yeast strains expressing wild type and mutant L-CPTI
Mitochondria (150 µg of protein) from the yeast strains expressing wild type L-CPTI, arginine, and glutamate substitution mutations were assayed for CPT activity and malonyl-CoA sensitivity as described under "Experimental Procedures." The results are the means ± S.D. of at least three independent experiments with different mitochondrial preparations.

Kinetic Characteristics of Mutant L-CPTIs-- Mutants E603D and E603Q exhibited normal saturation kinetics when the carnitine concentration was varied relative to a constant second substrate, palmitoyl-CoA (Fig. 3A), a property identical to that of the wild type L-CPTI. The kinetic characteristics of mutants R601A and R606A with respect to carnitine and palmitoyl-CoA were as reported in our previous publication (18). For mutants E603D, E603Q, and R606A, the calculated Km values for carnitine were similar to the wild type as shown in Table III and Ref. 18. However, the Vmax for carnitine for the E603Q mutant was 10-fold lower compared with the wild type and the E603D mutant, indicating a major effect of the mutation on catalytic activity. The catalytic efficiency as estimated by Vmax/Km for E603Q and E603D was decreased by 92.3 and 43.1%, respectively. With respect to the second substrate, palmitoyl-CoA, mutants E603D and E603Q exhibited normal saturation kinetics similar to the wild type (Fig. 3B) when the molar ratio of palmitoyl-CoA to albumin was fixed at 6.1:1. The calculated Km values for mutants E603D and E603Q were 3- and 7-fold lower, and the Vmax values were 62 and 93.7% lower than the wild type, respectively. For the E603Q, the catalytic efficiency was 40.7% lower than the wild type, but the E603D mutant exhibited catalytic efficiency similar to the wild type. Thus, substitution of the conserved Glu-603 residue with glutamine and the conserved Arg-601 and Arg-606 residues with alanine (18) caused a substantial loss in catalytic activity and malonyl-CoA sensitivity. In contrast, a conservative substitution of Glu-603 with aspartate significantly lowered malonyl-CoA sensitivity but had a minor effect on catalytic activity.


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Fig. 3.   Kinetic analysis of wild type and mutant L-CPTI activities. Isolated mitochondria (150 µg of protein) from the yeast strains expressing the wild type (), E603Q (black-diamond ), and E603D (black-triangle) mutants were assayed for CPT activity in the presence of increasing concentrations of carnitine (A) and palmitoyl-CoA (C). B and D, expanded dose-response curves for the E603Q mutant.


                              
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Table III
Kinetic characteristics of yeast-expressed wild type and mutant L-CPTIs
Isolated mitochondria (150 µg of protein) from the yeast strains expressing the wild type and glutamate substitution mutations were assayed for CPT activity in the presence of increasing concentrations of carnitine or palmitoyl-CoA. Values are averages of two independent experiments with different mitochondrial preparations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our site-directed mutagenesis studies of conserved residues in the C-terminal region of L-CPTI demonstrated that substitution of Arg-601 and Arg-606 with alanine resulted in almost complete loss in activity and a significant decrease in malonyl-CoA sensitivity. Within the C-terminal peptide sequence -RTETVR- (Fig. 1), the two arginine residues are highly conserved in the family of acyltransferases, and the other four residues are also conserved. Glu-603 is a conserved residue within the family of CPT enzymes, while other acyltransferases have aspartate at this position. A change of Glu-603 to alanine or histidine resulted in complete loss in L-CPTI activity. Substitution of the highly conserved Glu-590 with alanine did not have a major effect on catalytic activity (data not shown). The site-directed mutagenesis study described here is aimed at elucidating the function of these conserved acidic and basic residues found at the proximity of the active site of L-CPTI.

To determine the role of the conserved Glu-603 on catalysis and malonyl-CoA sensitivity, we separately changed the Glu-603 residue to alanine, histidine, glutamine, and aspartate (E603A, E603H, E603Q, and E603D, respectively) and determined the effect of the mutations on L-CPTI activity and malonyl-CoA sensitivity in the yeast-expressed mutant enzyme. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity. Since a conservative substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity, the presence of this negatively charged conserved residue Glu-603, is probably crucial for maintaining the configuration of the L-CPTI active site. The mutant L-CPTI with a replacement of Glu-603 with aspartate (E603D) or glutamine (E603Q) showed a 15-fold decrease in malonyl-CoA sensitivity, while a change of Arg-601 or Arg-606 to alanine resulted in an over 40-fold decrease in malonyl-CoA inhibition of the mutant L-CPTI (18). The loss in catalytic activity observed with the mutant L-CPTI (E603Q, E603D, R601A, and R606A) was in each case associated with a decrease in malonyl-CoA sensitivity, suggesting that these three conserved residues may be important for substrate and inhibitor binding. The effect of the mutations on activity and malonyl-CoA sensitivity suggests that this region of L-CPTI spanning the conserved C-terminal residues -RTETVR- may be important for the substrate (palmitoyl-CoA) and the inhibitor (malonyl-CoA) binding, probably through the common CoA moiety present in both compounds. Although there was a substantial decrease in Vmax for both substrates (carnitine and palmitoyl-CoA) with the E603D, E603Q, and R606A mutants (18), all the mutants showed an increase in the affinity for palmitoyl-CoA but not carnitine, suggesting that these residues are involved in binding palmitoyl-CoA and malonyl-CoA but not carnitine. This region of the protein may thus be involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or a low affinity acyl-CoA binding site.

As a rate-limiting enzyme that transports long chain fatty acids from the cytosol to the mitochondrial matrix, L-CPTI in the presence of carnitine catalyzes the conversion of long chain acyl CoAs to acylcarnitines (1, 2). Similar to other acyltransferases, L-CPTI contains a general acid/base, His-473, a highly conserved amino acid residue that may form a hydrogen bonding network or a salt bridge to a nearby conserved glutamate residue such as Glu-603 (23). We hypothesize that substitution of Glu-603 with alanine or histidine may disrupt a hydrogen bonding network or a salt bridge, perhaps to the highly conserved residue His-473 that is predicted to be at the active site of L-CPTI. The significantly reduced stability of the E603Q mutant implicates Glu-603 in the maintenance of active site architecture, suggesting substitution of Glu-603 with glutamine may also disrupt a hydrogen bonding network or a salt bridge to a residue like His-473 at the active site of L-CPTI. Furthermore, the site-directed mutagenesis studies demonstrate that even a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, suggesting that a change of Glu-603 to aspartate may result in the carboxylate being outside the hydrogen bond distance of His-473. Glu-603 may thus be required for L-CPTI stability and positioning of the imidazole ring of His-473 for efficient catalysis and inhibition, thus facilitating productive interaction with the substrates and the inhibitor (23). Mutation of the corresponding conserved residue in CPTII, Glu-500, to alanine resulted in 50% loss in activity (24).

In this report, we demonstrate that the conserved Glu-603 of L-CPTI is required for the structural stability of the enzyme. Despite its similar size and potential for hydrogen bonding formation, a glutamine residue cannot substitute for glutamate, suggesting that the negative charge of Glu-603 and/or its ability to serve as a strong hydrogen bond acceptor is needed for optimal catalysis, maintenance of active site integrity, and malonyl-CoA inhibition and binding. The reduced L-CPTI activity and malonyl-CoA sensitivity observed with the E603D mutant suggest that the loss of a methyl group may result in the carboxylate being outside the hydrogen bond distance of the conserved amino acid residue His-473, the predicted general acid/base at the active site. This suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and malonyl-CoA sensitivity. Also, since Glu-603 is located adjacent to Arg-601 and Arg-606, the negatively charged Glu-603 may be positioned to form hydrogen bonds and/or a salt bridge with the positively charged residues Arg-601 and Arg-606 that form the predicted CoA binding pocket. Disruption of the hydrogen bonding network or salt bridge may also cause loss of catalytic activity and malonyl-CoA sensitivity. Since only a 15-42-fold loss in malonyl-CoA sensitivity was observed in these mutants compared with the more than 100-fold loss in inhibitor sensitivity that we reported with the N-terminal residue mutations, it is predicted that this C-terminal region may constitute the low affinity malonyl-CoA binding site in L-CPTI (16). Characterization of the wild type and R601A, E603A, E603H, E603Q, E603D, and R606A mutant enzymes has led to the identification of conserved residues in the C-terminal region of L-CPTI that are important for catalytic activity and malonyl-CoA sensitivity. Since a mutation of any of these three conserved C-terminal residues (Arg-601, Glu-603, and Arg-606) substantially decreased catalytic activity and malonyl-CoA sensitivity, it is hypothesized that these residues are the major contact sites between L-CPTI and the CoA moiety of the substrate (palmitoyl-CoA) and the inhibitor (malonyl-CoA) and constitute the putative low affinity acyl-CoA/malonyl-CoA binding site in L-CPTI.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL52571 and by the American Heart Association-Northwest Affiliate (to G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Environmental and Biomolecular Systems, OGI School of Science and Engineering, 20000 N.W. Walker Rd., Beaverton, OR 97006-8921. Tel.: 503-748-1676; Fax: 503-748-1464; E-mail: gwoldeg@bmb.ogi.edu.

Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M210566200

    ABBREVIATIONS

The abbreviations used are: CPTI, carnitine palmitoyltransferase I; DCCD, dicyclohexylcarbodiimide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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