Biomedical subjects

D Linder

Publications and source records attributed to D Linder.

At least 55 records · Page 3Linked to original sources

PubMed1995

Trypsin-resistant protease activation mutants of an influenza virus.

New classes of mutants of influenza virus A/seal/Mass/1/80 are described in which the haemagglutinins (HA) have lost their protease cleavability by trypsin, but can be activated by elastase, chymotrypsin or thermolysin in different cell types. The same proteases that were required for activation of infectivity of the mutants also activated haemolysis and cell-fusing properties. The protease activation (pa)-mutants were non-pathogenic for chickens, but induced a protective immune response against a highly pathogenic challenge virus. The failure of the mutants to be activated by trypsin, but instead to be activated by the other proteases employed, was related to amino acid exchanges around the HA cleavage site. The cleavability of the chymotrypsin and elastase pa-mutants is most likely determined by replacement of Arg-1 by neutral amino acids such as Ile, Thr, Met or Leu, depending on the substrate specificity of the activating proteases. Cleavage activation of the thermolysin pa-mutants, on the other hand, became possible by insertion of a single Leu residue at position 4 of the HA2 polypeptide, which compensates for the loss of the Gly residue at the N terminus of the fusion peptide due to thermolysin cleavage. The correction of the mutations in revertants confirmed the conclusions drawn from sequence analyses of the pa-mutants.

Amino Acid Sequence↗

PubMed1994

Evidence for domain structures of the trifunctional protein and the tetrafunctional protein acting in glyoxysomal fatty acid beta-oxidation.

In plant glyoxysomes, an enzyme activity responsible for a particular step in the fatty acid beta-oxidation is located on more than one protein species. Various monofunctional enzymes and two forms of a multifunctional protein are involved in the degradation of cis-unsaturated fatty acids. delta 3, delta 2-Enoyl-CoA isomerase activity, previously found to be located on a monofunctional dimeric protein, is attributable to one form of the monomeric multifunctional protein (MFP). The presence or absence of isomerase activity allows us to differentiate between the tetrafunctional 76.5-kDa isoform (MFP II) and the trifunctional 74-kDa isoform (MFP I) in cucumber (Cucumis sativus) cotyledons. Both MFP I and MFP II exhibited blocked N-terminal structures. MFP I and MFP II are distinguishable from each other by their susceptibility to limited proteolysis. A series of examples is presented describing the preparation of enzymically active proteolytic fragments. We demonstrate that both forms of the monomeric MFP are composed of domains separable from each other without loss of activity. By fragmentation of MFP I and subsequent chromatography, a 60-kDa peptide was purified retaining hydratase and epimerase activity but lacking dehydrogenase activity. In addition, a highly positively charged fragment was observed carrying solely dehydrogenase activity. From MFP II, a 36-kDa fragment with hydratase activity was characterized. An enzymically inactive 46-kDa fragment was prepared from MFP II and sequenced at its unblocked N-terminus.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1994

Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans.

Glutaconate coenzyme A-transferase (Gct) from Acidaminococcus fermentans consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N-termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encoding glutaconate CoA-transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to sigma 70-type promoters from Escherichia coli. Primer-extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)-2-hydroxyglutarate to crotonyl-CoA in the pathway of glutamate fermentation by A. fermentans. The genes gctAB were expressed together in E. coli. Cell-free extracts of a transformant E. coli strain contained glutaconate CoA-transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).

Amino Acid Sequence↗

PubMed1994

The heterodisulfide reductase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic of pyridine-nucleotide-dependent thioredoxin reductases.

The genes hdrA, hdrB and hdrC, encoding the three subunits of the iron-sulfur flavoprotein heterodisulfide reductase, have been cloned and sequenced. HdrA (72.19 kDa) was found to contain a region of amino acid sequence highly similar to the FAD-binding domain of pyridine-nucleotide-dependent disulfide oxidoreductases. Additionally, 110 amino acids C-terminal to the FAD-binding consensus, a short polypeptide stretch (VX2CATID) was detected which shows similarity to the region of thioredoxine reductase that contains the active-site cysteine residues (VX2CATCD). These findings suggest that HdrA harbors the site of heterodisulfide reduction and that the catalytic mechanism of the enzyme is similar to that of pyridine-nucleotide-dependent thioredoxin reductase. HdrA was additionally found to contain four copies of the sequence motif CX2CX2CX3C(P), indicating the presence of four [4Fe-4S] clusters. Two such sequence motifs were also present in HdrC (21.76 kDa), the N-terminal amino acid sequence of which showed sequence similarity to the gamma-subunit of the anaerobic glycerol-3-phosphate dehydrogenase of Escherichia coli. HdrC is therefore considered to be an electron carrier protein that contains two [4Fe-4S] clusters. HdrB (33.46 kDa) did not show sequence similarity to other known proteins, but appears to possess a C-terminal hydrophobic alpha-helix that might function as a membrane anchor. Although hdrB and hdrC are juxtaposed, these genes are not near hdrA.

Amino Acid Sequence↗

PubMed1994

F420H2: quinone oxidoreductase from Archaeoglobus fulgidus. Characterization of a membrane-bound multisubunit complex containing FAD and iron-sulfur clusters.

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, was found to contain a membrane-bound F420H2: quinone oxidoreductase complex presumed to be involved in energy conservation during growth on lactate plus sulfate. After solubilization with dodecyl-beta-D-maltoside the complex was purified 32-fold with a yield of 24%. Using both gel filtration and native PAGE, an apparent molecular mass of approximately 270 kDa was determined. SDS/PAGE revealed the presence of at least seven polypeptides with apparent molecular masses 56, 45, 41, 39, 37, 33, and 32 kDa. The purified complex contained 1.6 mol FAD, 9 mol non-heme iron and 7 mol acid-labile sulfur/mol complex. It did not contain cytochromes, which were, however, present in the membrane fraction of A. fulgidus (3 nmol/mg membrane protein). The purified F420H2: quinone oxidoreductase complex catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone (apparent Km 190 microM) with reduced coenzyme F420 (apparent Km 50 microM) exhibiting a specific activity of 500 U/mg (apparent Vmax) at pH 8.0 (pH optimum) and 65 degrees C (temperature optimum). 2-Methyl-1,4-naphthoquinone (menadione), 2-hydroxy-1,4-naphthoquinone, 1,4-naphthoquinone, 2,3-dimethoxy-5-methyl-1,4- benzoquinone, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-ubiquinone) were also reduced with F420H2, albeit with lower rates. The physiological electron acceptor of the F420H2: quinone oxidoreductase complex is most likely the menaquinone found in the membrane fraction of A. fulgidus.

Amino Acid Sequence↗

PubMed1994

A single point mutation results in A allele-specific exon skipping in the bovine alpha s1-casein mRNA.

Bovine alpha s1-casein (alpha s1-CN) allele A is found in low allelic frequencies among different cattle breeds and is known to be characterized by the deletion of amino-acid residues 14 to 26 of the mature protein (as defined via the most common allele B), and a corresponding deletion of 39 bp from its cDNA. Based upon the genomic sequence of bovine alpha s1-CN [Koczan et al., Nucleic Acids Res. 19 (1991) 5591-5596], this allelic deviation can be interpreted as an absence of exon 4 from the A allele mRNA and protein product. We demonstrate that this allelic aberration is not caused by a genomic deletion across the exon-4 DNA, but is correlated with a single point mutation at position +6 in the splice donor sequence distal of exon 4, which results in upstream exon skipping during the serial splice reactions of the A allele alpha s1-CN pre-mRNA. The A-allele-specific mutation at position +6 is able to interrupt the perfect complementarity of the intron-4 splice donor signal (positions one to eight) with U1-snRNA, which may then no longer be able to compensate for a rather weak exon-4 upstream splice acceptor sequence in facilitating the initial binding of U2 auxiliary factor/65-kDa (U2AF65) to that polypyrimidine tract. This interpretation of the exon skipping mechanism in alpha s1-CN allele A is in agreement with similar results obtained [Hoffmann and Grabowski, Genes Dev. 6 (1992) 2554-2568] in an analysis of the rat preprotachykinin-encoding gene and in vitro experiments.

Alleles↗

PubMed1994

Cloning, sequencing and expression of the gene encoding the coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri in Escherichia coli.

The coenzyme B12 (adenosylcobalamin)-dependent 2-methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri. (a) The mgm gene encoding 2-methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8-kDa protein of 614 amino acids. It shows significant similarity in its C-terminal part to that of other cobamide-dependent enzymes. Probably, this is the coenzyme-binding region. (b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/PAGE and Western-blot analysis with rabbit antiserum directed against the native mutase. (c) Cell-free extracts from E. coli carrying the mgm gene showed 2-methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B12. Experiments are presented which suggest that the expression product is an apoenzyme.

Amino Acid Sequence↗

PubMed1994

Tungstate can substitute for molybdate in sustaining growth of Methanobacterium thermoautotrophicum. Identification and characterization of a tungsten isoenzyme of formylmethanofuran dehydrogenase.

Methanobacterium thermoautotrophicum (strain Marburg) was found to grow on media supplemented with tungstate rather than with molybdate. The Archaeon then synthesized a tungsten iron-sulfur isoenzyme of formylmethanofuran dehydrogenase. The isoenzyme was purified to apparent homogeneity and shown to be composed of four different subunits of apparent molecular masses 65 kDa, 53 kDa, 31 kDa, and 15 kDa and to contain per mol 0.4 mol tungsten, < 0.05 mol molybdenum, 8 mol non-heme iron, 8 mol acid-labile sulfur and molybdopterin guanine dinucleotide. Its molecular and catalytic properties were significantly different from those of the molybdenum isoenzyme characterized previously. The two isoenzymes also differed in their metal specificity: the active molybdenum isoenzyme was only synthesized when molybdenum was available during growth whereas the active tungsten isoenzyme was also generated during growth of the cells on molybdate medium. Under the latter conditions the tungsten isoenzyme was synthesized containing molybdenum rather than tungsten.

Aldehyde Oxidoreductases↗

PubMed1994

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase.

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m = 59 kDa/subunit), containing 2 +/- 0.2 mol FAD, 13.6 +/- 0.8 mol Fe and 10.8 +/- 1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.

Amino Acid Sequence↗

PubMed1994

Litomosoides carinii microfilarial sheaths: partial amino acid sequences of several major polypeptide constituents.

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

Amino Acid Sequence↗

PubMed1994

The influence of storage on cytotoxic drug activity in an ATP-based chemosensitivity assay.

The use of viability assays to assess the effect of antineoplastic agents on cell lines and tumor cells is an important investigative tool and may have clinical relevance. Such assays require very small quantities of drugs and it is the practice of many laboratories to freeze aliquots of drugs for use in these assays as required. We have investigated the stability of 11 different agents in an ATP-based chemosensitivity assay which is being evaluated for clinical use. The results show that most drugs maintain their biological activity well when frozen at -20 degrees C for periods up to 24 months, or occasionally at room temperature. However, 4-hydroperoxycyclophosphamide and mitomycin C are exceptions to this rule, and should not be kept frozen for more than 2-3 months. Cisplatin is unstable when frozen and then thawed, but maintained activity at room temperature for at least 6 months. Since biological activity may not correlate completely with chemical stability, further studies on the effect of storage are required, but it seems unlikely that the appropriate use of frozen aliquots is a major source of error in tumor chemosensitivity assays.

Adenosine Triphosphate↗

PubMed1994

Structural elements in glycoprotein 70 from polytropic Friend mink cell focus-inducing virus and glycoprotein 71 from ecotropic Friend murine leukemia virus, as defined by disulfide-bonding pattern and limited proteolysis.

The disulfide-bonding pattern of glycoprotein 70 (gp70), the surface glycoprotein (SU) encoded by the envelope gene of polytropic Friend milk cell focus-inducing virus, was elucidated and compared with that of glycoprotein 71 (gp71), the corresponding glycoprotein of the ecotropic Friend murine leukemia virus, which had previously been determined (M. Linder, D. Linder, J. Hahnen, H.-H. Schott, and Stirm, Eur. J. Biochem. 203:65-73, 1992). In the carboxy-terminal constant domain, in which these glycoproteins have about 97% sequence homology, the location of the four disulfide bonds was found to be analogous. In the amino-terminal differential domain, with about 37% sequence homology, 8 of the 12 cysteine residues of the ecotropic SU are conserved in the polytropic SU. In this domain, a similar clustering of disulfide bonds was detected, which led to the identification of three distinct disulfide-bonded regions in both glycoproteins. However, because of deletions and sequence deviations, the glycoproteins must have significantly different three-dimensional structures in these regions. Since the receptor-binding functions of both glycoproteins have been attributed to their amino-terminal domains and since each binds to a different receptor, these disulfide-bonded structures are likely candidates for receptor-binding functions. Limited proteolysis of both glycoproteins with various endoproteinases led to the identification of preferential proteolytic sites between disulfide-bonded regions, at the beginning of the hypervariable proline-rich region, and between differential and constant domains, further confirming the structural organization of the folded glycoproteins.

Amino Acid Sequence↗

PubMed1993

Pseudomonas aeruginosa cytotoxin-binding protein in rabbit erythrocyte membranes. An oligomer of 28 kDa with similarity to transmembrane channel proteins.

Rabbit erythrocyte membrane glycosylated 28-kDa protein was investigated in the membrane-bound as well as in the soluble state on an example of a Pseudomonas aeruginosa cytotoxin-binding component. When membranes were treated with trypsin/N-glycosidase F, a 13.5-kDa-binding active peptide residue is obtained as revealed by a ligand-blot technique after separation by SDS/PAGE under reducing conditions and electrophoretic transfer to nitrocellulose. Target-size analysis of intact membranes by radiation inactivation using 2-450 kGy gave a value of 29, 40 and 60 kDa for the binding-protein structure. This suggests that the native form of the binding peptide is associated as an oligomer. As seen in ligand-blot technique, 125I-cytotoxin binds with high affinity to water-channel integral protein CHIP28 from human erythrocyte membranes. The 20 N-terminal amino acids of the deglycosylated rabbit cytotoxin-binding protein show high similarity to transmembrane channel-like proteins.

Amino Acid Sequence↗

PubMed1993

L-serine and L-threonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups.

L-Serine dehydratase from the Gram-positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89-94]. It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram-positive bacterium, Clostridium propionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids. L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa). The purified enzyme exhibited a specific activity of 5 mu kat/mg protein and a Km for L-threonine of 7.7 mM. L-Serine (Km = 380 mM) was also deaminated, the V/Km ratio, however, being 118-fold lower than the one for L-threonine. L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate. L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for L-serine (Km = 5 mM) and was inhibited by L-cysteine (Ki = 0.5 mM) and D-serine (Ki = 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. L-Serine dehydratase was composed of two different subunits (alpha, m = 30 kDa; beta, m = 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from P. asaccharolyticus. Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichia coli was detected.

Amino Acid Sequence↗

PubMed1993

A novel very small subunit of a selenium containing [NiFe] hydrogenase of Methanococcus voltae is postranslationally processed by cleavage at a defined position.

A coenzyme-F420 non-reducing [NiFe] hydrogenase was isolated from Methanococcus voltae. It consists of three subunits. They are the products of the previously identified genes vhuA, vhuG and vhuU. The vhuU gene product is of only 25 amino acids. This novel very small hydrogenase subunit contains selenocysteine within a conserved amino-acid sequence previously shown to be involved in Ni coordination. The subunit is shorter than the predicted primary gene product and is therefore apparently post-translationally processed.

Amino Acid Sequence↗

PubMed1993

Identification of novel phosphorylation sites in murine A-type lamins.

We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and microsequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A-type lamins at the non-alpha-helical amino- and carboxy-terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A-type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis-specific phosphorylation at the cdc2-kinase-specific phosphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.

Amino Acid Sequence↗

PubMed1993

Purification and properties of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanobacterium thermoautotrophicum.

N5-Methyltetrahydromethanopterin:coenzyme M meth-yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy-conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purified 30-fold to apparent homogeneity. The purified enzyme had an apparent molecular mass of 670 kDa and was composed of seven different polypeptides of 34 kDa, 28 kDa, 24 kDa, 23 kDa, 21 kDa, 13 kDa, and 12 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native 670-kDa enzyme was found to contain 7.6 mol 5-hydroxybenzimidazolyl cobamide/mol, 37 mol non-heme iron/mol and 34 mol acid-labile sulfur/mol. Cobalt analyses after sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that the corrinoid was bound to the 23-kDa polypeptide. The apparent molecular masses of the polypeptides given above were determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without boiling the samples prior to analysis. When the samples were boiled, as is usually done, the 23-kDa polypeptide changed its apparent molecular mass to 33 kDa and the 21-kDa, 24-kDa, and 28-kDa polypeptides formed aggregates. The specific activity (apparent Vmax) of the purified methyltransferase preparation was 11.6 mumol.min-1.mg protein-1. The apparent Km for N5-methyltetrahydromethanopterin was 260 microM and that for coenzyme M was 60 microM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5-2-fold.

Amino Acid Sequence↗

PubMed1993

N5,N10-methenyltetrahydromethanopterin cyclohydrolase from the extremely thermophilic sulfate reducing Archaeoglobus fulgidus: comparison of its properties with those of the cyclohydrolase from the extremely thermophilic Methanopyrus kandleri.

Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83 degrees C and 98 degrees C, respectively. Both Archaea contain an active N5,N10-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus. The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of K2HPO4 and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of K2HPO4 (1 M) for optimal thermostability at 90 degrees C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high K2HPO4 concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed.

Amino Acid Sequence↗