Amino acid residues that determine functional specificity of NADP- and NAD-dependent isocitrate and isopropylmalate dehydrogenases (original) (raw)
Related papers
European Journal of Biochemistry, 1997
In a previous study we reported on the successful inversion of coenzyme specificity in isocitrate dehydrogenase (IDH) from NADP to NAD [Chen, R., A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity, Proc. Natl Acad. Sci. USA 92, 11 666Ϫ11 670]. Here, we explore alternative means to generate NAD dependence in the NADPdependent scaffold of Escherichia coli IDH. The results reveal that engineering a preference for NAD is constrained by the architecture of the IDH coenzyme binding pocket and confirms that the substituted Asp344 in the engineered enzyme is the major determinant of coenzyme specificity. Mutations in the 316Ϫ325 loop, which forms part of the coenzyme binding site, reduce activity through transmission of long-range conformational changes into the active site some 14 Å distant. Conformational changes seen upon substituting Cys332→Tyr are not directly involved with improving activity. Replacements at Cys201 reveal that subtle changes in the packing of hydrophobic residues (Met and Ile versus Leu) can elicit markedly different responses. We caution against using sequence alignments as the sole guide for mutagenesis and show how a combination of rational design of active-site residues based on X-ray structures and random substitutions at surrounding residues provides an efficient means to improve enzyme preference and catalytic efficiency towards novel substrates.
Proteins: Structure, Function, and Bioinformatics, 2007
Isocitrate dehydrogenase (ICDH) is an enzyme in the Krebs cycle that catalyses the dehydrogenation and subsequent decarboxylation of isocitrate to a-ketoglutarate and CO 2 using NAD þ or NADP þ as a cofactor. 1-3 ICDH evolved early and is widely distributed among archaea, bacteria, and eukarya. Such an evolutional background is reflected in diverse primary structures, various oligomeric states, and even different cofactor specificity. 4 ICDH and isopropylmalate dehydrogenase (IPMDH) form a family of homo-dimeric decarboxylating dehydrogenases, which share a unique cofactor-binding site that differs from the Rossmann fold found in many other dehydrogenases. 5-7 Based on a wealth of structural and enzymological information accumulated on these enzymes showing a strong preference for either NAD þ or NADP þ , the family of the dehydrogenases has become a target of many design efforts to change the preference. It has been reported that multiple mutations of seven residues at the cofactor-binding site successfully switched the specificity of Ec-ICDH from a 7000-fold preference for NADP þ over NAD þ to a 200-fold preference for NAD þ over NADP þ . 8 Remarkably, the overall activity of the mutant enzyme was comparable to that of wild-type NAD þ -dependent enzymes. Structural analyses revealed the interaction between the introduced Asp-344 residue and the vicinal hydroxyl groups of the adenosine ribose moiety of NAD þ . The multiple mutations also succeeded in shifting the binding site for the adenine ring and altering the ribose ring conformation from C3 0 -endo to C2 0 -endo puckering. 8 Naturally occurring NAD þ -specific ICDH thus attracted our interest for comparative studies with engineered NAD þ -specific ICDH and NAD þ -specific IPMDHs. We chose ICDH from an aerobic chemolithotrophic bacterium, Acidithiobacillus thiooxidans, 9 for the present structural study. NAD þ -specific ICDHs have been found in some chemolithotrophic bacteria that possess a 2-oxoglutarate dehydrogenase-deficient Krebs cycle. 10,11 It has been proposed that NAD þ -specific ICDH may be reminiscent of an enzyme that functions in CO 2 fixation in an Abbreviations: ICDH, isocitrate dehydrogenase; IPMDH, isopropylmalate dehydrogenase.
Structure, 1994
Background: The leucine biosynthetic enzyme 3-isopropylmalate dehydrogenase (IMDH) belongs to a unique class of bifunctional decarboxylating dehydrogenases. The two best-known members of this family, IMDH and isocitrate dehydrogenase (IDH), share a common structural framework and catalytic mechanism but have different substrate and cofactor specificities. IMDH is NAD+-dependent, while IDHs occur in both NAD+-dependent and NADP+-dependent forms. Results: We have co-crystallized Thermus thermophilus IMDH with NAD + and have determined the structure at 2.5 A resolution. NAD + binds in an extended conformation. Comparisons with the structure in the absence of cofactor show that binding induces structural changes of up to 2.5 A in the five loops which form the dinucleotide-binding site. The adenine and diphosphate moieties of NAD+ are bound via interactions which are also present in the NADP+-IDH complex. Amino acids which interact with the NADP + 2'-phosphate in IDH are substituted or absent in IMDH. The adenosine ribose forms two hydrogen bonds with Asp278, and the nicotinamide and nicotinamide ribose interact with Glu87 and Asp78, all unique to IMDH. Conclusions: NAD+ binding induces a conformational transition in IMDH, resulting in a structure that is intermediate between the most 'open' and 'closed' decarboxylating dehydrogenase conformations. Physiological specificity of IMDH for NAD + versus NADP + can be explained by the unique interaction between Asp278 and the free 2'-hydroxyl of the NAD + adenosine, discrimination against the presence of the 2'-phosphate by the negative charge on Asp278, and the absence of potential favorable interactions with the 2'-phosphate of NADP + .
Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase
Proceedings of the National Academy of Sciences, 1989
The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylmalate dehydrogenase. Isocitrate dehydrogenase contains an unusual clasp-like domain in which both polypeptide chains in the dimer interlock. Based on the structure of isocitrate dehydrogenase and conservation with isopropylmalate dehydrogenase, we suggest that the active site lies in an interdomain pocket close to the phosphorylation site.
Functional relevance of dynamic properties of Dimeric NADP-dependent Isocitrate Dehydrogenases
BMC Bioinformatics, 2012
Background Isocitrate Dehydrogenases (IDHs) are important enzymes present in all living cells. Three subfamilies of functionally dimeric IDHs (subfamilies I, II, III) are known. Subfamily I are well-studied bacterial IDHs, like that of Escherischia coli. Subfamily II has predominantly eukaryotic members, but it also has several bacterial members, many being pathogens or endosymbionts. subfamily III IDHs are NAD-dependent. The eukaryotic-like subfamily II IDH from pathogenic bacteria such as Mycobacterium tuberculosis IDH1 are expected to have regulation similar to that of bacteria which use the glyoxylate bypass to survive starvation. Yet they are structurally different from IDHs of subfamily I, such as the E. coli IDH. Results We have used phylogeny, structural comparisons and molecular dynamics simulations to highlight the similarity and differences between NADP-dependent dimeric IDHs with an emphasis on regulation. Our phylogenetic study indicates that an additional subfamily (IV...
Journal of Structural Biology
Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to a-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotidebinding domain have been linked to NAD + vs. NADP + specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD + -specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD + in Domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD + binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD + vs. NADP + recognition.
Journal of Molecular Biology, 2000
Isocitrate dehydrogenase catalyses the two step, acid base, oxidative decarboxylation of isocitrate to a-ketoglutarate. Lysine 230 was suggested to act as proton donor based on geometry and spatial proximity to isocitrate. To clarify further the role of lysine 230, we co-crystallized the lysine-to-methionine mutant (K230M) with isocitrate and with a-ketoglutarate. Crystals were¯ash-frozen and the two structures were determined and re®ned to 2.1 A Ê. Several new features were identi®ed relative to the wild-type structure. Seven side-chains previously unplaced in the wildtype structure were identi®ed and included in the model, and the amino acid terminus was extended by an alanine residue. Many additional water molecules were identi®ed. Examination of the K230M active sites (K230M isocitrate and K230Mketoglutarate) revealed that tyrosine 160 protrudes further into the active site in the presence of either isocitrate or a-ketoglutarate in K230 M than it does in the wild-type structure. Also, methionine 230 was not as fully extended, and asparagine 232 rotates $30 toward the ligand permitting polar interactions. Outside the active site cleft a tetragonal volume of density was identi®ed as a sulfate molecule. Its location and interactions suggest it may in¯uence the equilibrium between the tetragonal and the orthorhombic forms of isocitrate dehydrogenase. Differences observed in the active site water structure between the wild-type and K230M structures were due to a single point mutation. A water molecule was located in the position equivalent to that occupied by the wild-type e-amine of lysine 230; a water molecule in that location in K230M suggests it may in¯uence catalysis in the mutant. Comparison of K230M complexed with isocitrate and a-ketoglutarate illuminates the in¯uence a ligand has on active site water structure.
PLOS ONE, 2016
Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD + and NADP + as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP + shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP + , but the same residues formed no observable interactions in the case of NAD +. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the k cat /K M value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP + , only R50 makes a contribution (about-1 kcal/mol) to NAD + binding. In the absence of both positive charges the enzyme was unable to discriminate NADP + from NAD +. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP +-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD + in the case of the NADP +-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.
Crystal structure of 3-isopropylmalate dehydrogenase in complex with NAD+ and a designed inhibitor
Bioorganic & Medicinal Chemistry, 2009
Isopropylmalate dehydrogenase (IPMDH) is the third enzyme specific to leucine biosynthesis in microorganisms and plants, and catalyzes the oxidative decarboxylation of (2R,3S)-3-isopropylmalate to a-keto isocaproate using NAD + as an oxidizing agent. In this study, a thia-analogue of the substrate was designed and synthesized as an inhibitor for IPMDH. The analogue showed strong competitive inhibitory activity with K i = 62 nM toward IPMDH derived from Thermus thermophilus. Moreover, the crystal structure of T. thermophilus IPMDH in a ternary complex with NAD + and the inhibitor has been determined at 2.8 Å resolution. The inhibitor exists as a decarboxylated product with an enol/enolate form in the active site. The product interacts with Arg 94, Asn 102, Ser 259, Glu 270, and a water molecule hydrogen-bonding with Arg 132. All interactions between the product and the enzyme were observed in the position associated with keto-enol tautomerization. This result implies that the tautomerization step of the thia-analogue during the IPMDH reaction is involved in the inhibition.