Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase

X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion. Only the metal-IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.     

Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.     

Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution.

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.     

Cold-adaptation mechanism of mutant enzymes of 3-isopropylmalate dehydrogenase from Thermus thermophilus

Random mutagenesis of Thermus thermophilus 3-isopropylmalate dehydrogenase revealed that a substitution of Val126Met in a hinge region caused a marked increase in specific activity, particularly at low temperatures, although the site is far from the binding residues for 3-isopropylmalate and NAD. To understand the molecular mechanism, residue 126 was substituted with one of eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile or Phe. Circular dichroism analyses revealed a decreased thermal stability of the mutants (Delta T ((1/2))= 0-13 degrees C), indicating structural perturbations caused by steric conflict with surrounding residues having larger side chains. Kinetic parameters, k(cat) and K(m) values for isopropylmalate and NAD, were also affected by the mutation, but the resulting k(cat)/K(m) values were similar to that of the wild-type enzyme, suggesting that the change in the catalytic property is caused by the change in free-energy level of the Michaelis complex state relative to that of the initial state. The kinetic parameters and activation enthalpy change (Delta H (double dagger)) showed good correlation with the van der Waals volume of residue 126. These results suggested that the artificial cold adaptation (enhancement of k(cat) value at low temperatures) resulted from the destabilization of the ternary complex caused by the increase in the volume of the residue at position 126.     

The effects of multiple ancestral residues on the Thermus thermophilus 3-isopropylmalate dehydrogenase

Previously, we showed that mutants of Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70 degrees C when compared with the wild-type IPMDH.     

Crystallization and preliminary X-ray data for 3-isopropylmalate dehydrogenase of Thermus thermophilus

The gene coding for 3-isopropylmalate dehydrogenase of Thermus thermophilus was cloned and expressed in Escherichia coli. The extracted enzyme was crystallized in a suitable size for X-ray crystallographic studies. The crystals have a space group of P3(1)21 or P3(2)21 with a = b = 78.6 A and c = 157.4 A.     

Novel substrate specificity of designer 3-isopropylmalate dehydrogenase derived from Thermus thermophilus HB8

Redesigning of an enzyme for a new catalytic reaction and modified substrate specificity was exploited with 3-isopropylmalate dehydrogenase (IPMDH). Point-mutation on Gly-89, which is not in the catalytic site but near it, was done by changing it to Ala, Ser, Val, and Pro, and all the mutations changed the substrate specificity. The mutant enzymes showed higher catalytic efficiency (kcat/Km) than the native IPMDH when malate was used as a substrate instead of 3-isopropylmalate. More interestingly, an additional insertion of Gly between Gly-89 and Leu-90 significantly altered the substrate-specificity, although the overall catalytic activity was decreased. Particularly, this mutant turned out to efficiently accept D-lactic acid, which was not accepted as a substrate by wild-type IPMDH at all. These results demonstrate the opportunity for creating nove,enzymes by modification of amino acid residues that do not directly participate in catalysis, or by insertion of additional residues.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.     

Crystallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HB8 chimeric 3-isopropylmalate dehydrogenase

A chimeric gene was constructed by fusing the Bacillus subtilis and Thermus thermophilus genes coding for 3-isopropylmalate dehydrogenase, and expressed in Escherichia coli. The chimeric enzyme was crystallized in a size suitable for X-ray structure analysis. The crystal has a space group of P3(1)21 or P3(2)21, a = b = 77.1 A and c = 158.3 A, which is isomorphous with that of the native enzyme from T. thermophilus.     

Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method.

We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-172-->Val substitution in all of the mutants. The thermal stability of the thermophile IPMDH was improved by introducing the amino acid substitution.     

Isolation of a gene encoding 3-isopropylmalate dehydrogenase from rice

3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85) is a key enzyme in the biosynthesis of leucine and glucosinolates in plants. IPMDH is a bifunctional dimeric enzyme that catalyzes dehydrogenation and decarboxylation reactions in the presence of NAD⁺. The leuB gene encoding IPMDH has been identified in a variety of bacteria and some plants. In this study, we analyze the gene for IPMDH from Oryza sativa (OsIPMDH). The analysis of an EST sequence and rice genome revealed a full-length open reading frame encoding 389 amino acids that correspond to a protein of approximately 41.2 kD. The predicted amino acid sequence of OsIPMDH was highly homologous with the IPMDHs from plants and bacteria. The OsIPMDH expression analysis in a leuB mutant of Escherichia coli revealed that OsIPMDH was capable of functionally complementing the leuB mutant. These results indicate that OsIPMDH encodes for a protein of 3-isopropylmalate dehydrogenase in rice.     

Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus

A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase (IPMDH) from a random mutant library was a double mutant containing the mutations I11V and S92F that were found in cold-adapted mutant IPMDHs previously isolated. To elucidate the effect of each mutation on enzymatic activity, I11V and six multiple mutant IPMDHs were constructed and analyzed. All of the multiple mutant IPMDHs were found to be improved in catalytic activity at moderate temperatures by increasing the k(cat) with a simultaneous increase of K(m) for the coenzyme NAD(+). k(cat) was improved by a decrease in the activation enthalpy, DeltaH( not equal). The multiple mutants did not show large reduction in thermal stability, and one of them showed enhanced thermal stability. Mutation from I11 to V was revealed to have a stabilizing effect. Mutants showed increased thermal stability when the mutation I11V was combined. This indicates that it is possible to construct mutants with enhanced thermal stability by combining stabilizing mutation. No additivity was observed for the thermodynamic properties of catalytic reaction in the multiple mutant IPMDHs, implying that the structural changes induced by the mutations were interacting with each other. This indicates that careful and detailed tuning is required for enhancing activity in contrast to thermal stability.     

Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus

A leuB strain of Thermus thermophilus TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50 degrees C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50 degrees, 60 degrees, 62 degrees, 65 degrees, 67 degrees, and 70 degrees C, step by step. All the mutant strains except for one isolated at 50 degrees C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Alal 12Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.     

High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low DeltaC(p) of unfolding.

To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change DeltaG degrees of each unfolding process. Other thermodynamic parameters, enthalpy change DeltaH, entropy change DeltaS and heat capacity change DeltaC(p), were derived from the temperature dependence of DeltaG degrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaG degrees on temperature resulting from a low DeltaC(p). The thermophilic IPMDH had a significantly lower DeltaC(p), 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low DeltaC(p) of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area DeltaASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature T(s). The DeltaG degrees maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature T(m) is not necessarily accompanied by a higher DeltaG degrees maximum.     

Studies on interdomain interaction of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus, by constructing chimeric enzymes

In our previous study, we showed that a chimeric isopropylmalate dehydrogenase, 2T2M6T, between an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, isopropylmalate dehydrogenases (the name roughly denotes the primary structure; the first 20% from the N-terminal is coded by the thermophile leuB gene, next 20% by mesophile, and the rest by the thermophile gene) denatured in two steps with a stable intermediate, suggesting that in the chimera some of the interdomain interaction was lost by amino acid substitutions in the "2M" part. To identify the residues involved in the interdomain interactions, the first and the second halves of the 2M part of the chimera were substituted with the corresponding sequence of the thermophile enzyme. Both chimeras, 3T1M6T and 2T1M7T, apparently showed one transition in the thermal denaturation without any stable intermediate state, suggesting that the cooperativity of the conformational stability was at least partly restored by the substitutions. The present study also suggested involvement of one or more basic residues in the unusual stability of the thermophile enzyme. Received: September 29, 1998 / Accepted: June 25, 1999     

Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from thermus thermophilus

Cytochrome c oxidase is a respiratory enzyme catalysing the energy-conserving reduction of molecular oxygen to water. The crystal structure of the ba(3)-cytochrome c oxidase from Thermus thermophilus has been determined to 2.4 A resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa. A structure-based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g. a new electron transfer pathway leading directly from Cu(A) to Cu(B). Specific features of the ba(3)-oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O(2-), OH(-) or H(2)O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c. New aspects of the proton pumping mechanism could be identified.     

Crystallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HB8 chimeric 3-isopropylmalate dehydrogenase and thermostable mutants of it.

A new type of chimeric 3-isopropylmalate dehydrogenase (2T2M6T) was produced by expressing the fused gene of Bacillus subtilis and Thermus thermophilus. The enzyme shows heat stability intermediate between those of the parents. The crystal of the enzyme belongs to the space group of P3(2)21, with cell dimensions of a = b = 78.9 A and c = 158.9 A. Two thermostable mutants of the chimeric enzyme were prepared by site-directed mutagenesis and then crystallized.     

Atomic level characterization of the nonproton ligand-sensing domain of ASIC3 channels

Acid-sensing ion channels (ASICs) are known to be primarily activated by extracellular protons. Recently, we characterized a novel nonproton ligand (2-guanidine-4-methylquinazoline, GMQ), which activates the ASIC3 channel subtype at neutral pH. Using an interactive computational-experimental approach, here we extend our investigation to delineate the architecture of the GMQ-sensing domain in the ASIC3 channels. We first established a GMQ binding mode and revealed that residues Glu-423, Glu-79, Leu-77, Arg-376, Gln-271, and Gln-269 play key roles in forming the GMQ-sensing domain. We then verified the GMQ binding mode using ab initio calculation and mutagenesis and demonstrated the critical role of the above GMQ-binding residues in the interplay among GMQ, proton, and Ca(2+) in regulating the function of ASIC3. Additionally, we showed that the same residues involved in coordinating GMQ responses are also critical for activation of the ASIC3(E79C) mutant by thiol-reactive compound DTNB. Thus, a range of complementary techniques provide independent evidence for the structural details of the GMQ-sensing domain at atomic level, laying the foundation for further investigations of endogenous nonproton ligands and gating mechanisms of the ASIC3 channels.     

The primary prostaglandin-inactivating enzyme of human placenta is a dimeric short-chain dehydrogenase

The native form of NAD-dependent 15-hydroxyprostaglandin dehydrogenase of human placenta has a mol. wt. of about 50 0002 while the subunit tool. wt. is around 2g 0002 suggesting a dimeric quaternary structure. These propertie% the amino acid composition, insensitivity to EDTA, and inhibition patterns show general similarities to other short-chain dehydrogenases. Several hormones tested did not influence the activity of 15-hydroxyprostaglandin dehydrogenase2 but an unusual activation by two anti-depressant drugs was found and may relate to the existence of a natural regulatory factor.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .