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R Sterner

Publications and source records attributed to R Sterner.

At least 19 recordsLinked to original sources
PubMed2001

Imidazole glycerol phosphate synthase from Thermotoga maritima. Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex.

Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF with N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction.

Amino Acid Sequence↗
PubMed2001

Stability, catalytic versatility and evolution of the (beta alpha)(8)-barrel fold.

The (beta alpha)(8)-barrel is a versatile single-domain protein fold that is adopted by a large number of enzymes. The (beta alpha)(8)-barrel fold has been used as a model to elucidate the structural basis of protein thermostability and in studies to interconvert catalytic activities or substrate specificities by rational design or directed evolution. Recently, the (beta alpha)(4)-half-barrel was identified as a possible structural subdomain.

Catalysis↗
PubMed2001

Dissection of a (betaalpha)8-barrel enzyme into two folded halves.

The (betaalpha)8-barrel, which is the most frequently encountered protein fold, is generally considered to consist of a single structural domain. However, the X-ray structure of the imidazoleglycerol phosphate synthase (HisF) from Thermotoga maritima has identified it as a (betaalpha) 8-barrel made up of two superimposable subdomains (HisF-N and HisF-C). HisF-N consists of the four N-terminal (betaalpha) units and HisF-C of the four C-terminal (betaalpha) units. It has been postulated, therefore, that HisF evolved by tandem duplication and fusion from an ancestral half-barrel. To test this hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purified and characterized. Separately, HisF-N and HisF-C are folded proteins, but are catalytically inactive. Upon co-expression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalytically fully active HisF-NC complex. These findings support the hypothesis that the (betaalpha)8-barrel of HisF evolved from an ancestral half-barrel and have implications for the folding mechanism of the members of this large protein family.

Amino Acid Sequence↗
PubMed2001

Thermophilic adaptation of proteins.

Hyperthermophilic organisms optimally grow close to the boiling point of water. As a consequence, their macromolecules must be much more thermostable than those from mesophilic species. Here, proteins from hyperthermophiles and mesophiles are compared with respect to their thermodynamic and kinetic stabilities. The known differences in amino acid sequences and three-dimensional structures between intrinsically thermostable and thermolabile proteins will be summarized, and the crucial role of electrostatic interactions for protein stability at high temperatures will be highlighted. Successful attempts to increase the thermostability of proteins, which were either based on rational design or on directed evolution, are presented. The relationship between high thermo-stability of enzymes from hyperthermophiles and their low catalytic activity at room temperature is discussed. Not all proteins from hyperthermophiles are thermostable enough to retain their structures and functions at the high physiological temperatures. It will be shown how this shortcoming can be surpassed by extrinsic factors such as large molecular chaperones and small compatible solutes. Finally, the potential of thermostable enzymes for biotechnology is discussed.

Adaptation, Biological↗
PubMed2001

Divergent evolution of (betaalpha)8-barrel enzymes.

The (betaalpha)8-barrel is the most versatile and most frequently encountered fold among enzymes. It is an interesting question how the contemporary (betaalpha)8-barrels are evolutionarily related and by which mechanisms they evolved from more simple precursors. Comprehensive comparisons of amino acid sequences and three-dimensional structures suggest that a large fraction of the known (betaalpha)8-barrels have divergently evolved from a common ancestor. The mutational interconversion of enzymatic activities of several (betaalpha)8-barrels further supports their common evolutionary origin. Moreover, the high structural similarity between the N- and C-terminal (betaalpha)4 units of two (betaalpha)8-barrel enzymes from histidine biosynthesis indicates that the contemporary proteins evolved by tandem duplication and fusion of the gene of an ancestral 'half-barrel' precursor. In support of this hypothesis, recombinantly produced 'half-barrels' were shown to be folded, dimeric proteins.

Biological Evolution↗
PubMed2000

Structural evidence for evolution of the beta/alpha barrel scaffold by gene duplication and fusion.

The atomic structures of two proteins in the histidine biosynthesis pathway consist of beta/alpha barrels with a twofold repeat pattern. It is likely that these proteins evolved by twofold gene duplication and gene fusion from a common half-barrel ancestor. These ancestral domains are not visible as independent domains in the extant proteins but can be inferred from a combination of sequence and structural analysis. The detection of subdomain structures may be useful in efforts to search genome sequences for functionally and structurally related proteins.

Aldose-Ketose Isomerases↗
PubMed2000

Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways.

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.

Aldose-Ketose Isomerases↗
PubMed2000

Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima.

BACKGROUND: Oligomeric proteins may have been selected for in hyperthermophiles because subunit association provides extra stabilization. Phosphoribosylanthranilate isomerase (PRAI) is monomeric and labile in most mesophilic microorganisms, but dimeric and stable in the hyperthermophile Thermotoga maritima (tPRAI). The two subunits of tPRAI are associated back-to-back and are locked together by a hydrophobic loop. The hypothesis that dimerization is important for thermostability has been tested by rationally designing monomeric variants of tPRAI. RESULTS: The comparison of tPRAI and PRAI from Escherichia coli (ePRAI) suggested that levelling the nonplanar dimer interface would weaken the association. The deletion of two residues in the loop loosened the dimer. Subsequent filling of the adjacent pocket and the exchange of polar for apolar residues yielded a weakly associating and a nonassociating monomeric variant. Both variants are as active as the parental dimer but far more thermolabile. The thermostability of the weakly associating monomer increased significantly with increasing protein concentration. The X-ray structure of the nonassociating monomer differed from that of the parental subunit only in the restructured interface. The orientation of the original subunits was maintained in a crystal contact between two monomers. CONCLUSIONS: tPRAI is dimeric for reasons of stability. The clearly separated responsibilities of the betaalpha loops, which are involved in activity, and the alphabeta loops, which are involved in protein stability, has permitted the evolution of dimers without compromising their activity. The preserved interaction in the crystal contacts suggests the most likely model for dimer evolution.

Aldose-Ketose Isomerases↗
PubMed1999

Efficient expression, purification and crystallisation of two hyperthermostable enzymes of histidine biosynthesis.

Enzymes from hyperthermophiles can be efficiently purified after expression in mesophilic hosts and are well-suited for crystallisation attempts. Two enzymes of histidine biosynthesis from Thermotoga maritima, N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase and the cyclase moiety of imidazoleglycerol phosphate synthase, were overexpressed in Escherichia coli, both in their native and seleno-methionine-labelled forms, purified by heat precipitation of host proteins and crystallised. N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase crystallised in four different forms, all suitable for X-ray structure solution, and the cyclase moiety of imidazoleglycerol phosphate synthase yielded one crystal form that diffracted to atomic resolution. The obtained crystals will enable the determination of the first three-dimensional structures of enzymes from the histidine biosynthetic pathway.

Aldose-Ketose Isomerases↗
PubMed1999

Balance training for persons with functionally unstable ankles.

STUDY DESIGN: A nonrandomized 2-group pretest-posttest design. OBJECTIVES: To determine the effects of a 4-week balance training program during stance on a single leg. BACKGROUND: Individuals who have experienced multiple episodes of inversion ankle sprains often participate in balance training programs. Balance training is performed to treat existing proprioceptive deficits and to restore ankle joint stability, presumably by retraining altered afferent neuromuscular pathways. The effectiveness of such programs on individuals with functionally unstable ankles has yet to be established. METHODS AND MEASURES: Prior to and following training, subjects with self-reported functionally unstable ankles (5 women and 8 men, mean age = 21.9 +/- 3.1 years) and nonimpaired subjects (6 women and 7 men, mean age = 21.2 +/- 2.5 years) completed a static balance assessment for both limbs as well as the ankle joint functional assessment tool questionnaire (AJFAT). The subjects from both groups participated in a unilateral, multilevel, static and dynamic balance training program 3 times a week for 4 weeks. Subjects from the experimental group trained only the involved limb, and the nonimpaired group trained a randomly selected limb. A stability index (SI) was calculated during the balance assessment to indicate the amount of platform motion. Compared to low stability indices, high stability indices indicate greater platform motion during stance and therefore less stability. RESULTS: Following training, subjects from both groups demonstrated significant improvements in balance ability. When balance was assessed at a low resistance to platform tilt (stability level 2), the posttraining scores of both the subjects with unstable ankles (mean SI = 2.63 +/- 1.92) and the nonimpaired subjects (mean SI = 2.69 +/- 2.32) were significantly better than their pretraining scores (mean SIs = 5.93 +/- 3.65 and 4.67 +/- 3.43, respectively). Assessed at a high resistance to platform tilt (stability level 6), the posttraining scores of both subjects with unstable ankles (mean SI = 1.27 +/- 0.66) and the nonimpaired subjects (mean SI = 1.37 +/- 0.66) were significantly better than their pretraining scores (mean SIs = 2.30 +/- 1.88 and 2.04 +/- 1.43, respectively). Additionally, the posttraining AJFAT scores of subjects with unstable ankles (25.78 +/- 3.80) and the nonimpaired subjects (29.15 +/- 5.27) were significantly greater than their pretraining scores (17.11 +/- 3.44 and 22.92 +/- 5.22, respectively), indicating an overall improvement in perceived ankle joint functional stability. CONCLUSIONS: This study suggests that balance training is an effective means of improving joint proprioception and single-leg standing ability in subjects with unstable and nonimpaired ankles.

Adolescent↗
PubMed1998

A histidine gene cluster of the hyperthermophile Thermotoga maritima: sequence analysis and evolutionary significance.

The sequences of histidine operon genes in hyperthermophiles are informative for understanding high protein thermostability and the evolution of metabolic pathways. Therefore, a cluster of eight his genes from the hyperthermophilic and phylogenetically early bacterium Thermotoga maritima was cloned and sequenced. The cluster has the gene order hisDCBdHAFI-E, lacking only hisG and hisBp, and does not contain intercistronic regions. This compact organization of his genes resembles the his operon of enterobacteria. Sequence analysis downstream of the stop codon of hisI-E identifies a region with a significantly higher cytosine over guanosine content, which is indicative of a rho-dependent termination of transcription of the his operon. Multiple sequence alignments of N1-((5'-phosphoribosyl)-formimino)-5-aminoimidazole-4-carboxyam ide ribonucleotide isomerase (HisA) and of the cycloligase moiety of imidazoleglycerol phosphate synthase (HisF) support the previous assignment of the (beta alpha)8-barrel fold to these proteins. The alignments also reveal a second phosphate-binding motif located in the first halves of both enzymes and thereby support the hypothesis that HisA and HisF have evolved by a sequence of two gene duplication events. Comparison of the amino acid compositions of HisA and HisF from mesophiles and thermophiles shows that the thermostable variants of both enzymes contain a significantly increased number of charged amino acid residues and may therefore be stabilized by additional salt bridges.

Aldose-Ketose Isomerases↗
PubMed1997

Ferredoxin from the hyperthermophile Thermotoga maritima is stable beyond the boiling point of water.

Heat-stable proteins from hyperthermophilic microorganisms are ideally suited for investigating protein stability and evolution. We measured with differential scanning calorimetry and optical absorption spectroscopy the thermal stability of [4Fe-4S] ferredoxin from Thermotoga maritima (tfdx), which is a small electron transfer protein. The results are consistent with two-state unfolding at the record denaturation temperature of 125 degrees C. According to the crystal structure at 1.75 A resolution, T. maritima ferredoxin contains a significantly increased number of hydrogen bonds that involve charged amino acid side-chains, compared to thermolabile ferredoxins. Thus, our results suggest that polar interactions substantially contribute to protein stability at very high temperatures. Moreover, because small [4Fe-4S] ferredoxins seem to have occurred early in evolution, the extreme thermostability of tfdx supports the hypothesis that life originated at high temperatures.

Calorimetry, Differential Scanning↗
PubMed1997

Crystal structure at 2.0 A resolution of phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima: possible determinants of protein stability.

The structural basis of thermostability of proteins is of great scientific and biotechnological interest. Differences in the X-ray structues of orthologous proteins from hyperthermophilic and mesophilic organisms can indicate crucial stabilizing interactions. To this end the crystal structure of dimeric phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima (tPRAI) was determined using phases derived from the isomorphous replacement method and was refined at 2.0 A resolution. The comparison to the known 2.0 A structure of PRAI from Escherichia coli (ePRAI) shows that tPRAI has the complete TIM- or (beta alpha)8-barrel fold, whereas helix alpha5 in ePRAI is replaced by a loop. The subunits of tPRAI associate via the N-terminal faces of their central beta-barrels. Two long, symmetry-related loops that protrude reciprocally into cavities of the other subunit provide for multiple hydrophobic interactions. Moreover, the side chains of the N-terminal methionines and the C-terminal leucines of both subunits are immobilized in a hydrophobic cluster, and the number of salt bridges is increased in tPRAI. These features appear to be mainly responsible for the high thermostability of tPRAI. In contrast to other hyperthermostable enzymes, tPRAI at 25 degrees C is catalytically more efficient than ePRAI, mainly due to its small K(M) value for the substrate [Sterner, R., Kleemann, G. R., Szadkowski, H., Lustig, A., Hennig, M., & Kirschner, K. (1996) Protein Sci. 5, 2000-2008]. The increased number of hydrogen bonds between the phosphate ion and tPRAI compared to ePRAI could be responsible for this effect.

Aldose-Ketose Isomerases↗
PubMed1996

Small structural changes account for the high thermostability of 1[4Fe-4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima.

BACKGROUND: The characterization of the structural features that account for the high thermostability of some proteins is of great scientific and biotechnological interest. Proteins from hyperthermophilic organisms with optimum growth temperatures of 80 degrees C and higher generally show high intrinsic stabilities. The comparison of high resolution X-ray structures of these proteins with their counterparts from mesophilic organisms has therefore helped to identify potentially stabilizing forces in a number of cases. Small monomeric proteins which comprise only a single domain, such as ferredoxins, are especially suitable for such comparisons since the search for determinants of protein stability is considerably simplified. RESULTS: The 1.75 A crystal structure of the extremely thermostable 1[4Fe-4S] ferredoxin from Thermotoga maritima (FdTm) was determined and compared with other monocluster-containing ferredoxins with different degrees of thermostability. CONCLUSIONS: A comparison of the three-dimensional structure of FdTm with that of ferredoxins from mesophilic organisms suggests that the very high thermostability of FdTm is unexpectedly achieved without large changes of the overall protein structure. Instead, an increased number of potentially stabilizing features is observed in FdTm, compared with mesophilic ferredoxins. These include stabilization of alpha helices, replacement of residues in strained conformation by glycines, strong docking of the N-terminal methionine and an overall increase in the number of hydrogen bonds. Most of these features stabilize several secondary structure elements and improve the overall rigidity of the polypeptide backbone. The decreased flexibility will certainly play a relevant role in shielding the iron-sulfur cluster against physiologically high temperatures and further improve the functional integrity of FdTm.

Amino Acid Sequence↗
PubMed1996

Small-angle X-ray scattering reveals differences between the quaternary structures of oxygenated and deoxygenated tarantula hemocyanin.

Small-angle X-ray scattering (SAXS) curves have been recorded for the oxygenated and deoxygenated states of the 4 x 6-meric hemocyanin from the tarantula Eurypelma californicum. A comparison of the curves shows that the quaternary structures of the two states are different by three criteria, which all indicate that the hemocyanin is less compact in the oxygenated compared to the deoxygenated form: (a) The radius of gyration is 8.65 +/- 0.05 nm for the deoxy- and 8.80 +/- 0.05 nm for the oxy-form. (b) The maximum particle dimension amounts to 25.0 +/- 0.5 nm for the deoxy- and to 27.0 +/- 0.5 nm for the oxy-form. (c) A dip in the intramolecular distance distribution function p(r) is more pronounced and shifted to larger distances in the oxy-form. The p(r) functions based on SAXS measurements were compared to p(r) functions deduced from published electron microscopical images of three different 4 x 6-meric hemocyanins from closely related species. The p(r) functions of SAXS and electron microscopy were similar in one case, whereas in the other two cases the distance between the two 12-meric half-molecules had to be changed by 1-1.5 nm to obtain good agreement. The differences between the p(r) functions of oxygenated and deoxygenated 4 x 6-meric tarantula hemocyanin are much larger than one would expect from a comparison of X-ray structures of the oxygenated and deoxygenated states of a closely related 6-meric hemocyanin. Thus, the conformational changes upon oxygenation occur at various levels of the quaternary structure, as postulated by hierarchical theories of allosteric interactions.

Animals↗
PubMed1996

An NMR-derived model for the solution structure of oxidized Thermotoga maritima 1[Fe4-S4] ferredoxin.

The solution structure of the 60-residue 1[Fe4-S4] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1[Fe4-S4] ferredoxins from Desulfovibrio gigas and Desulfovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the T. maritima protein. In particular, the T. maritima protein displayed a beta-sheet structure made up of strands located at the very NH(2) and COOH termini of the protein, and an internal alpha-helix. The internal beta-sheet observed in the D. gigas and D. africanus ferredoxins could not be confirmed in T. maritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure.

Amino Acid Sequence↗