Ju-Yi Hsieh

Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)+-dependent Malic Enzyme FUNCTIONAL ROLES OF GLUTAMINE 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The ΔG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.

Functional roles of the tetramer organization of malic enzyme

Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In addition, the enzyme has distinct active sites within each subunit. The mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) isoform behaves cooperatively and allosterically and exhibits a quaternary structure in dimer-tetramer equilibrium. The cytosolic NADP+-dependent malic enzyme (c-NADP-ME) isoform is noncooperative and nonallosteric and exists as a stable tetramer. In this study, we analyze the essential factors governing the quaternary structure stability for human c-NADP-ME and m-NAD(P)-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME and m-NAD(P)-ME. Size distribution analysis demonstrated that human c-NADP-ME exists mainly as a tetramer, whereas human m-NAD(P)-ME exists as a mixture of dimers and tetramers. Kinetic data indicated that the enzyme activity of c-NADP-ME is not affected by disruption of the interface. There are no significant differences in the kinetic properties between AB and AD dimers, and the dimeric form of c-NADP-ME is as active as tetramers. In contrast, disrupting the interface of m-NAD(P)-ME causes the enzyme to be less active than wild type and to become less cooperative for malate binding; the kcat values of mutants decreased with increasing Kd,24 values, indicating that the dissociation of subunits at the dimer or tetramer interfaces significantly affects the enzyme activity. The above results suggest that the tetramer is required for a fully functional m-NAD(P)-ME. Taken together, the analytical ultracentrifugation data and the kinetic analysis of these interface mutants demonstrate the differential role of tetramer organization for the c-NADP-ME and m-NAD(P)-ME isoforms. The regulatory mechanism of m-NAD(P)-ME is closely related to the tetramer formation of this isoform.

Structural basis of antizyme-mediated regulation of polyamine homeostasis

Significance Polyamines are small organic compounds that carry multiple positive charges at physiological pH. With a high capacity to interact with the acidic surface patches of proteins and nucleic acids, polyamines may regulate a variety of cellular processes, and the fluctuations in the intracellular polyamine levels are rigorously controlled during cell growth and differentiation through the interplay between the enzyme ornithine decarboxylase (ODC) and two regulatory proteins: antizyme (Az) and antizyme inhibitor (AzIN). ODC initiates the polyamine biosynthetic pathway, whereas Az decreases polyamine concentrations by both inhibiting ODC activity and channeling ODC for proteolytic degradation. AzIN neutralizes Az function to restore polyamine levels. Here we provide the long-sought structural information and previously unidentified functional insights into this delicate regulatory circuit. Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN–Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

Influential factor contributing to the isoform-specific inhibition by ATP of human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of the nucleotide binding site Lys346.

Human mitochondrial NAD(P)+‐dependent malic enzyme (m‐NAD‐ME) is a malic enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleotide‐binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m‐NAD‐ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m‐NAD‐ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant enzyme displays a shift in its cofactor preference from NAD+ to NADP+ upon increasing kcat,NADP and decreasing Km,NADP. Furthermore, the cooperative binding of malate becomes less significant in human m‐NAD‐ME after mutation of Lys346. The h value for the wild‐type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m‐NAD‐ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the enzyme to be much less sensitive to ATP, similar to cytosolic NADP‐dependent malic enzyme. Substitution of Lys to Arg did not change the isoform‐specific inhibition of the enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild‐type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.

Minimal Antizyme Peptide Fully Functioning in the Binding and Inhibition of Ornithine Decarboxylase and Antizyme Inhibitor

Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC50: 0.20 µM) similar to that of AZ-95-228 (IC50: 0.16 µM), even though a large segment spanning residues 177–228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC50 values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC50 values comparable to that of AZ_WT and formed AZ-ODC complexes with K d,AZ-ODC values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function.

Engineering of the cofactor specificities and isoform-specific inhibition of malic enzyme

Malic enzyme (ME) is a family of enzymes that catalyze a reversible oxidative decarboxylation of l-malate to pyruvate with simultaneous reduction of NAD(P)+ to NAD(P)H. According to the cofactor specificity, the mammalian enzyme can be categorized into three isoforms. The cytosolic (c) and mitochondrial (m) NADP+-dependent MEs utilize NADP+ as the cofactor. The mitochondrial NAD(P)+-dependent ME can use either NAD+ or NADP+ as the cofactor. In addition, the m-NAD(P)-ME isoform can be inhibited by ATP and allosterically activated by fumarate. In this study, we delineated the determinants for cofactor specificity and isoform-specific inhibition among the ME isoforms. Our data strongly suggest that residue 362 is the decisive factor determining cofactor preference. All the mutants containing Q362K (Q362K, K346S/Q362K, Y347K/Q362K, and K346S/Y347K/Q362K) have a larger kcat,NADP value compared with the kcat,NAD value, indicating that the enzyme has changed to use NADP+ as the preferred cofactor. Furthermore, we suggest that Lys-346 in m-NAD(P)-ME is crucial for the isoform-specific ATP inhibition. The enzymes containing the K346S mutation (K346S, K346S/Y347K, K346S/Q362K, and K346S/Y347K/Q362K) are much less inhibited by ATP and have a larger Ki,ATP value. Kinetic analysis also suggests that residue 347 functions in cofactor specificity. Here we demonstrate that the human K346S/Y347K/Q362K m-NAD(P)-ME has completely shifted its cofactor preference to become an NADP+-specific ME. In the triple mutant, Lys-362, Lys-347, and Ser-346 work together and function synergistically to increase the binding affinity for NADP+.

Functional role of fumarate site Glu59 involved in allosteric regulation and subunit–subunit interaction of human mitochondrial NAD(P)+‐dependent malic enzyme

Here we report on the role of Glu59 in the fumarate‐mediated allosteric regulation of the human mitochondrial NAD(P)+‐dependent malic enzyme (m‐NAD‐ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, KA,Fum and αKA,Fum. The E59D enzyme, although retaining the allosteric property, is quite different from the wild‐type in enzyme activation. The KA,Fum and αKA,Fum of E59D are also much greater than those of the wild‐type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild‐type and E59Q enzymes exist as a dimer–tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer–tetramer model similar to that of the wild‐type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate‐binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m‐NAD‐ME from mitochondrial NADP+‐dependent malic enzyme and cytosolic NADP+‐dependent malic enzyme in fumarate activation and malate cooperativity.

Structural characteristics of the nonallosteric human cytosolic malic enzyme

Human cytosolic NADP(+)-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in Km,NADP, Km,malate, and kcat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.

Determinants of Nucleotide-Binding Selectivity of Malic Enzyme

Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD+-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP+ as the cofactor without a significant increase in catalysis using NAD+ as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k cat,NAD, suggesting that His at residue 362 may be more beneficial than Gln for NAD+ binding. Furthermore, the S346I/K347D/K362H mutant had a very large K m,NADP value compared to other mutants, suggesting that this mutant exclusively utilizes NAD+ as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K m,NAD values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the K m values for NAD+ and NADP+ of the quadruple mutants were significantly decreased, while either k cat,NAD or k cat,NADP was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD+-utilizing enzymes by a considerable increase in catalysis using NAD+ as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding affinity of NAD+. Here, we demonstrate that a series of E314A-containing c-NADP-ME quadruple mutants have been changed to NAD+-utilizing enzymes by abrogating NADP+ binding and increasing NAD+ binding.

Dual roles of Lys(57) at the dimer interface of human mitochondrial NAD(P)+-dependent malic enzyme.

Human m-NAD(P)-ME [mitochondrial NAD(P)+-dependent ME (malic enzyme)] is a homotetramer, which is allosterically activated by the binding of fumarate. The fumarate-binding site is located at the dimer interface of the NAD(P)-ME. In the present study, we decipher the functional role of the residue Lys57, which resides at the fumarate-binding site and dimer interface, and thus may be involved in the allosteric regulation and subunit-subunit interaction of the enzyme. In the present study, Lys57 is replaced with alanine, cysteine, serine and arginine residues. Site-directed mutagenesis and kinetic analysis strongly suggest that Lys57 is important for the fumarate-induced activation and quaternary structural organization of the enzyme. Lys57 mutant enzymes demonstrate a reduction of Km and an elevation of kcat following induction by fumarate binding, and also display a much higher maximal activation threshold than WT (wild-type), indicating that these Lys57 mutant enzymes have lower affinity for the effector fumarate. Furthermore, mutation of Lys57 in m-NAD(P)-ME causes the enzyme to become less active and lose co-operativity. It also increased K0.5,malate and decreased kcat values, indicating that the catalytic power of these mutant enzymes was significantly impaired following mutation of Lys57. Analytical ultracentrifugation analysis demonstrates that the K57A, K57S and K57C mutant enzymes dissociate predominantly into dimers, with some monomers present, whereas the K57R mutant forms a mixture of dimers and tetramers, with a small amount of the enzyme in monomeric form. The dimeric form of these Lys57 mutants, however, cannot be reconstituted into tetramers with the addition of fumarate. Modelling structures of the Lys57 mutant enzymes show that the hydrogen bond network in the dimer interface where Lys57 resides may be reduced compared with WT. Although the fumarate-induced activation effects are partially maintained in these Lys57 mutant enzymes, the mutant enzymes cannot be reconstituted into tetramers through fumarate binding and cannot recover their full enzymatic activity. In the present study, we demonstrate that the Lys57 residue plays dual functional roles in the structural integrity of the allosteric site and in the subunit-subunit interaction at the dimer interface of human m-NAD(P)-ME.