Search PubMed⌕ Search

Biomedical subjects

T K Sundaram

Publications and source records attributed to T K Sundaram.

At least 19 recordsLinked to original sources
PubMed1996

Tetrameric malate dehydrogenase from a thermophilic Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and characterization of the recombinant enzyme.

The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid. The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa. Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species. Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs. MDHs and LDHs resemble each other in catalytic mechanism and several other respects. However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs. The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric. A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E. coli plasmid vector. BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E. coli strain carrying the clone. MDH purified from BI and that purified from the E. coli strain carrying the MDH gene clone appear to be identical proteins by several criteria. A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters. Whereas the MDH is a stable tetramer in the pH range 5-7, it appears to be converted into a stable dimer at pH 3.5. This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH.

Amino Acid Sequence↗
PubMed1995

Catalytic-rate improvement of a thermostable malate dehydrogenase by a subtle alteration in cofactor binding.

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2'- and 3'-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.

Aspartic Acid↗
PubMed1992

Overexpression of the Thermus aquaticus B malate dehydrogenase-encoding gene in Escherichia coli.

Expression of the Thermus aquaticus B malate dehydrogenase (MDH)-encoding gene (mdh), cloned in Escherichia coli, was initially at a relatively low level (0.1% of soluble cell protein) and was effected by read-through from the tac promoter in the plasmid vector used. An enhancement in expression to 0.4% of soluble cell protein was achieved by shortening the intervening sequence between the promoter and the translation start codon of mdh. An NdeI restriction site (5'-CAT-ATG-3') was engineered in the shortened fragment, which also changed the start codon from GTG to ATG. This resulted in an eightfold increase in expression, to 3.2% of soluble cell protein. Expression was further increased by subcloning the mdh gene via the engineered NdeI site, into two plasmid expression vectors, one carrying the E. coli trpP promoter and the other the E. coli mdhP promoter. In both these expression systems, 40-50% of the soluble cell protein was T. aquaticus MDH. This suggests that expression of the cloned T. aquaticus mdh in E. coli is enhanced predominantly by the optimisation of transcription and translation initiation signals. Moreover, the base composition of the coding region and the pattern of codon usage dictated by it appear to have little effect on expression. Heat treatment of the cell extract at 85 degrees C further effected purification of T. aquaticus MDH to over 80% of the soluble cell protein. The MDHs purified to homogeneity from the high-expression clones were identical with the MDH isolated from T. aquaticus B cells with respect to all measured parameters.

Base Sequence↗
PubMed1990

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B.

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.

Amino Acid Sequence↗
PubMed1989

Isocitrate dehydrogenase from thermophilic and mesophilic bacteria. Isolation and some characteristics.

1. Simple methods incorporating the principle of selective enzyme elution from a triazinyl dye adsorbent with a mixture of NADP+ and isocitrate are described for isolating NADP+-linked isocitrate dehydrogenase in pure state from several mesophilic and thermophilic bacteria. 2. Several characteristics of the isocitrate dehydrogenases have been examined, viz. molecular size, amino acid composition including the content of sulphydryl groups, thermostability and structural homology by the criterion of immunological cross-section.

Amino Acids↗
PubMed1989

Regulation of isocitrate dehydrogenase by phosphorylation in Escherichia coli K-12 and a simple method for determining the amount of inactive phosphoenzyme.

In several Escherichia coli K-12 strains grown on a limiting concentration of glucose, isocitrate dehydrogenase (IDH) was inactivated about 90% after cessation of growth upon exhaustion of the glucose. Such inactivation has been previously observed in several E. coli strains but not in E. coli K-12 (unless acetate was added to the bacterial culture when growth ceased). IDH was inactivated 75 to 80% in all E. coli K-12 strains we examined during growth on acetate. The inactivation involved phosphorylation of the enzyme and is considered to be a regulatory mechanism facilitating metabolite flow along the glyoxylate shunt. Phospho-IDH interacted with antibodies to enzymatically active IDH. We have devised a method, based on this immunological cross-reaction, for determining the proportions of active and inactive (phospho-) IDH in cell extracts.

Acetates↗
PubMed1988

Stability and immunological cross-reactivity of malate dehydrogenases from mesophilic and thermophilic sources.

The thermostability in vitro of dimeric and tetrameric malate dehydrogenases [S)-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mesophilic and thermophilic bacteria shows a good correlation to the growth temperature of the source organism but no consistent relationship to enzyme subunit structure. The thermophile malate dehydrogenases are, in general, more resistant to the surfactants, sodium dodecyl sulphate (SDS) and hexadecyltrimethylammonium bromide, and to the denaturants, guanidinium chloride and urea, than their mesophilic counterparts, with the dimer in each thermal class being more resistant to the chemical perturbants than the tetramer. Sedimentation analysis suggests that denaturation of the malate dehydrogenases by acid-periodate or SDS produces discrete subunits, whereas denaturation by guanidinium chloride followed by carboxymethylation yields ill-defined protein species. SDS and acid-periodate were therefore preferred to generate denatured malate dehydrogenases for use as immunogens and antigens. The native malate dehydrogenases exhibit immunological cross-reactivity only when they are in the same oligomeric form and derived from closely related species, which may, however, be from different thermal classes. Taking immunological cross-reactivity as an indicator of structural similarity, this supports the idea that the thermophilic trait evolved independently within each phyletic line. With denatured malate dehydrogenases as immunogens and antigens, cross-reactivity is manifested between all the malate dehydrogenases examined. This suggests that appreciable primary structural homology exists between the malate dehydrogenases, whether dimeric or tetrameric, from thermophiles and mesophiles and from various taxa.

Bacillus↗
PubMed1986

Action of surfactants on porcine heart malate dehydrogenase isoenzymes and a simple method for the differential assay of these isoenzymes.

The cationic surfactant, cetyl (hexadecyl) trimethylammonium bromide (CTAB), completely inactivates porcine heart cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) at concentrations (of surfactant) which do not affect the activity of the mitochondrial isoenzyme. These concentrations are close to, or higher than, the critical micelle concentration of CTAB. An increase in the ionic strength of the medium significantly retards the CTAB-induced inactivation of the cytoplasmic enzyme. The enzyme is also markedly protected against CTAB inactivation by NADH; L-malate on its own has no effect but a combination of NADH and L-malate affords greater protection than NADH alone. The CTAB inactivation is not reversed by dilution of the surfactant. The highly selective action of CTAB on the two malate dehydrogenases, which correlates well with their electrostatic charges, has been exploited for a simple and reliable differential assay of these isoenzymes. The anionic surfactant, sodium dodecyl sulphate (SDS), at concentrations well below the critical micelle concentration, inactivates both isoenzymes, but the mitochondrial enzyme is significantly more sensitive than its cytoplasmic counterpart. There is thus some correlation, though not as strong as with CTAB, between SDS inactivation and the charges of the two malate dehydrogenases. An increase in ionic strength has opposite effects on the two isoenzymes: the mitochondrial enzyme becomes more resistant and the cytoplasmic enzyme less so. Both isoenzymes are rendered more resistant to SDS by the inclusion of NADH. Inactivation of the enzymes caused by short exposure to SDS is largely reversed by dilution of the detergent, but longer exposure leads to progressive irreversible loss of activity. NADH very effectively protects the isoenzymes against irreversible inactivation. It is likely that a reversible phase of inactivation precedes an irreversible phase and that in the former phase SDS acts competitively with NADH. Both malate dehydrogenases possess considerable resistance to the nonionic detergent, Triton X-100.

Animals↗
PubMed1984

Malate dehydrogenases from actinomycetes: structural comparison of Thermoactinomyces enzyme with other actinomycete and Bacillus enzymes.

Malate dehydrogenases from bacteria belonging to the genus Thermoactinomyces are tetrameric, like those from Bacillus spp., and exhibit a high degree of structural homology to Bacillus malate dehydrogenase as judged by immunological cross-reactivity. Malate dehydrogenases from other actinomycetes are dimers and do not cross-react with antibodies to Bacillus malate dehydrogenase.

Bacillus↗
PubMed1980

Malate dehydrogenase from thermophilic and mesophilic bacteria. Molecular size, subunit structure, amino acid composition, immunochemical homology, and catalytic activity.

Malate dehydrogenases isolated from a number of mesophilic, moderately thermophilic, and extremely thermophilic bacteria yield upon denaturation subunits of molecular weight 32 000--36 000. Determination of their native molecular weights shows that some of the enzymes are dimeric and others are tetrameric; the two types are distributed in each of the three classes of bacteria. The amino acid compositions of the enzymes show no consistent trend that can be related to the progression of thermostability from the mesophile through the moderate thermophile to the extreme thermophile species. The tetrameric enzyme species all exhibit a high level of structural homology as judged by the criterion of immunological cross-reaction. Little cross-reaction occurs, however, between the tetramers and the dimers. The dimeric enzyme from the extreme thermophile, Thermus aquaticus, cross-reacts only weakly, if at all, even with dimeric malate dehydrogenases. The catalytic activities of the malate dehydrogenases vary over a wide range. Potassium chloride, organic solvents such as acetone, and the protein denaturants urea and guanidine hydrochloride activate a number of the malate dehydrogenases under the assay conditions employed. The diversity among the bacterial malate dehydrogenases, manifested not only in molecular size and subunit structure but also in properties such as catalytic activity and the dependence of this activity on electrolytes, organic solvents, and denaturants, indicates significant structural differences between several of these cognate enzyme species.

Amino Acids↗
PubMed1979

Regulatory characteristics of phosphoenolpyruvate carboxylase from the extreme thermophile, Thermus aquaticus.

Phosphoenolpyruvate carboxylase from the extremely thermophilic bacterium, Thermus aquaticus YT-1, exhibits a virtually absolute requirement for acetyl CoA and there is strong positive cooperativity in the interaction of this activator with the enzyme. Several tricarboxylic acid cycle intermediates inhibit the enzyme. These findings suggest an anaplerotic role for the enzyme and an allosteric modulation of its activity by acetyl CoA and tricarboxylic acid cycle intermediates.

Acetyl Coenzyme A↗
PubMed1979

Pyruvate carboxylase from a thermophilic Bacillus: some molecular characteristics.

Analysis of the native enzyme and of the subunits produced upon its denaturation shows that pyruvate carboxylase from a thermophilic Bacillus is a tetramer with a molecular weight (mean value) of 558,000 and that the four polypeptide subunits are probably identical. The three functions (carboxyl carrier, carboxylation, and carboxyl transfer) in the pyruvate carboxylation reaction must therefore reside in this quarter-molecular polypeptide. The enzyme molecule contains four atoms of zinc and four molecules of D-biotin, and in the electron microscope the disposition of its four subunits presents a rhombic appearance. Reaction of the denatured enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reveals 10 sulfhydryl groups/subunit. In the native enzyme less than one of these groups reacts with DTNB. By contrast, all of these groups (11/subunit) of the native chicken liver pyruvate carboxylase are accessible to DTNB. The thermophile enzyme is also more resistant to other sulfhydryl reagents and to denaturation under certain conditions than the avian enzyme.

Animals↗
PubMed1979

Simple efficient methods for the isolation of malate dehydrogenase from thermophilic and mesophilic bacteria.

Malate dehydrogenase from a number of bacteria drawn from several genera and representing the mesophilic, moderately thermophilic and extremely thermophilic classes was isolated by procedures which involve only a small number of steps (in most cases only two), of which the key one is affinity chromatography on 5'-AMP--Sepharose and/or on NAD+--hexane--agarose. Electrophoretic analysis of the native enzymes in polyacrylamide gel and of the denaturated enzymes in sodium dodecyl sulphate/polyacrylamide gel revealed no significant protein impurity in the purified preparations. The yields ranged from about 40% to over 80%. The malate dehydrogenases from the extreme thermophiles and from some of the moderate thermophiles are appreciably less efficient catalytically than their mesophilic homologues.

Adenosine Monophosphate↗
PubMed1978

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp.

Isocitrate lyase was isolated in homogeneous state from a thermophilic Bacillus. The enzyme has a mol.wt. of 180000 and a pI of 4.5 and contains threonine as the N-terminal residue. It resembles in size the cognate enzyme from the mesophilic bacterium Pseudomonas indigofera, but is smaller than the enzyme from the eukaryotic fungus Neurospora crassa. All three lyases are tetramers and similar in amino acid composition, but the thermophile enzyme is distinctive from its mesophilic coutnerparts in possessing a lower catalytic-centre activity, greater resistance to chemical and thermal denaturation and fewer thiol groups and in being strongly activated by salts. Salt activation, by 0.4M-KCl, is about 3-fold at 30 degrees C and pH 6.8 and weakens progressively as the temperature or pH is raised. The activation is probably due to a change in the enzyme conformation caused by the electrolyte modifying the interaction between charged groups or between hydrophobic groups in protein. The possible significance of the salt activation, of the relative paucity of thiol groups and of the greater resistance to chemical denaturants is discussed. Besides its effect on the Vmax., KCl produces large increases in the magnitude of several kinetic parameters. A rise in reaction temperature from 30 to 55 degrees C produces a somewhat similar result. In view of these peculiar features, the patterns of inhibition of enzyme activity by compounds such as succinate and phosphoenolpyruvate were examined at 30 and 55 degrees C in the presence and absence of KCl.

Amino Acids↗