SummaryA soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O 2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox þ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O 2 . Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O 2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O 2 in the regulation of pneumococcal transformability and virulence.

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The fast reaction if2 50 msec) observed previously in the refolding of thermally unfolded ribonuclease A (disulfide bonds intact) has now been studied by two properties indicative of enzyme function: binding of a competitive inhibitor (2'CMP) and hydrolysis of a substrate (CpA -C > p + A). Both the binding and catalytic reactions are fast (<2 msec) compared to refolding. Binding of 2'CMP occurs during both fast and slow refolding reactions, and the protein folded in the fast reaction has a normal binding constant for 2'CMP. Recovery of enzymatic activity during the fast refolding reaction, as measured by the rate of CpA hydrolysis, parallels the kinetic curve for 2'CMP binding. When the kinetics of refolding are measured by the burying of exposed tyrosi'ie groups, no difference is found. The presence of 2'CMP has no effect on the kinetics of refolding.The results show that the fast refolding reaction does not yield an intermediate in the refolding of RNase A. Instead, both fast and slow refolding reactions have a common product, fully active RNase A. Although they show a 100-fold difference in rates of refolding, the starting materials for the fast and slow refolding reactions have similar properties, as regards: (a) the molar absorbancy at 286 nm, reflecting the state of exposed tyrosine groups, and (b) their apparent failure to bind 2'CMP.Both fast and slow refolding reactions have been observed in studies of the reversible unfolding transitions of a few simple small proteins: in the pH-induced refolding of staphylococcal nuclease (1) and bovine pancreatic RNase A (2) and in the solvent-induced refolding of horse-heart ferricytochrome c (3, 4) and chicken egg-white lysozyme (5) in guanidine solutions. The interpretation of these fast refolding reactions has been in doubt, but it has been supposed that they represent the formation of one or more intermediates in refolding: either intermediates on the normal pathway (1) or abortive intermediates, not on the direct pathway (3, 4). An alternative possibility is that the fast reactions represent the conversion of intermediates to final product. For RNase A, this may be tested by measuring the enzymatic activity of the product of fast refolding in a time range where the extent of slow refolding is small.In previous work (2), the refolding of RNase A has been measured by the changes in absorbance at 286 nm or at 240 nm that result chiefly from shielding tyrosine groups from solvent. There are six tyrosine groups in RNase A. They are found in different regions of the three-dimensional structure (6, 7) rather remote from the active site, and all of the tyrosine groups are at least partly shielded from water at neutral pH, where they are not ionized (7). Titration experiments (8,9) show that three of the six tyrosine groups are not free to ionize normally in native RNase A, and spectral studies (10,11)

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H 2 O 2 oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) is an important species of lactic acid bacteria currently used in the industrial production of fermented milk products. L. delbrueckii subsp. bulgaricus is an aerotolerant anaerobe that obtains most of its energy from homolactic fermentation (12). It does not require strict anaerobic growth conditions and tolerates the concentration of O 2 in air. Even though L. delbrueckii subsp. bulgaricus does not use O 2 in its energetic metabolism, it is likely that the presence of oxygen in its environment can influence its physiology. Indeed, some lactic acid bacteria possess oxidases that utilize molecular oxygen to oxidize substrates such as pyruvate (22) or NADH (2,5,8,16,(23)(24)(25). As these oxidation reactions cannot occur under anaerobic conditions, metabolism in the presence of oxygen cannot be identical to that in the absence of oxygen. Also, the activities of these oxidases can produce partially reduced oxygen species such as the superoxide radical (O 2 .Ϫ ), hydrogen peroxide (H 2 O 2 ), the hydroxyl radical (HO . ), and other peroxyl radicals or peroxides that will cause an oxidative stress in the cell. It is therefore expected that the presence of oxygen will induce a specific cellular response to such oxidative stress. In this work, a difference in the growth of L. delbrueckii subsp. bulgaricus in the presence and absence of oxygen was observed. It was also found that L. delbrueckii subsp. bulgaricus could reduce oxygen into hydrogen peroxide with an NADH oxidase, probably to eliminate the oxygen present. However, this detoxification of oxygen led to an overproducti...

The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 250. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn-HCl) or urea, show that functional bovine pancreatic ribonuclease A (RNase A; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) is formed in both the fast and slow phases of the refolding process. We conclude that the guanidine-unfolded state of RNase A is an equilibrium mixture of fast-and slow-refolding species, as was found previously for the heat-unfolded state at low pH. The fraction of the fast-refolding species in guanidine or ureaunfolded RNase A is the same as that in the heat-unfolded protein at pH 2.Previous work has shown that the fast-refolding species disappears as the pH is raised from 3 to 5 for heat-unfolded RNase A. This pH effect is not present in refolding from concentrated GdnHCl solutions: the same proportion of the fast-refolding species is found from pH 2 to pH 6, and also from 2 M to 6 M Gdn-HCI at pH 5.8. We conclude that the same proportion of the fast-refolding species is present at equilibrium whenever the residual structure in unfolded RNase A is reduced to a low level, and that the structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain.The observed rates of protein folding reactions are many orders of magnitude faster than predicted from a purely random search of all possible configurations for a random coil polypeptide chain devoid of structuret. A possible explanation for this difference is that an unfolded protein is not a random coil. The elements of residual structure play a crucial role in directing the course of protein folding, and thus considerably restrict the possible pathways (1) There seems to be a contradiction between the two proposals: (a) that a guanidine-unfolded protein behaves as a homogeneous random chain and (b) that this same unfolded state still possesses some elements of residual structure which increase the rate and determine the pathway(s) of folding. However, these proposals are compatible if the residual structure, important for the folding process, is not present as such in the unfolded state, but is rapidly formed after refolding is initiated. For instance, formation of a-helical segments could take place within the dead time of stopped-flow measurements (6) and limit the possible pathways for refolding, if the a-helical segments are stable under the conditions in which refolding is initiated, without a requirement of prior slow steps in refolding to provide a stabilizing environment.Studies of the refolding of heat-unfolded RNase A (7-11) have shown that the heat-unfolded state does not behave as a single species in refolding. Instead, distinct fast-refolding and slow-refolding species of the heat-unfold...

Depending upon the conditions under which polymerization takes place, pure tubulin can assemble into microtubules following either the usual monotonic kinetics or a more complex oscillatory mechanism. When present, these oscillations involve large cyclic changes in the extent of polymer formed before a steady‐state is reached. Analysis of the microtubules formed at different times shows that these oscillations involve marked redistribution in both the length and number of microtubules. No significant difference is found between two populations of microtubules corresponding to the same level of assembly, one for which the extent of polymerization will remain stable with time and one for which it will decrease by as much as 90% in the next oscillation. The amplitude of these oscillations is sensitive to changes in the concentrations of protein, nucleotide (GTP, GDP or GMPpNp), magnesium ion or GTP regenerating system. A complete shift from an oscillatory to a monotonic polymerization can be induced by a minor increase in the concentration of free nucleotide, GTP or GDP.

of H-Ras bound to GDP (Milburn et al., 1990) and to the Gé rard Le Bras, Gisè le Le Bras, non-hydrolysable GTP analogues GPPNP (Pai et al., Isabelle Janoueix-Lerosey 2 , 1990) and GPPCP (Milburn et al., 1990) (Willumsen et al., 1986), and the recent crystal structure interactions (Nassar et al., 1995(Nassar et al., , 1996. This region probably constitutes a general docking site for the effectors The small G protein Rap2A has been crystallized in of small G proteins. The switch II region, on the other complex with GDP, GTP and GTPγS. The Rap2A-hand, is not essential for proper effector recognition, but GTP complex is the first structure of a small G protein may interact with guanine nucleotide exchange factors with its natural ligand GTP. It shows that the hydroxyl (reviewed in Polakis and McCormick, 1993). As most of group of Tyr32 forms a hydrogen bond with the these data were obtained for H-Ras, it remains essentially γ-phosphate of GTP and with Gly13. This interaction unsettled whether the numerous small G proteins of does not exist in the Rap2A-GTPγS complex. Tyr32 is the Ras family undergo the same GDP/GTP structural conserved in many small G proteins, which probably transition as H-Ras . also form this hydrogen bond with GTP. In addition,The H-Ras structures, by locating essential proteinTyr32 is structurally equivalent to a conserved arginine nucleotide interactions, have also been a reference in the that binds GTP in trimeric G proteins. The actual longstanding debate on how the GTPase activity of small participation of Tyr32 in GTP hydrolysis is not yet G proteins works (reviewed in Maegley et al., 1996). The clear, but several possible roles are discussed. The rate of hydrolysis of GTP is important for the duration of conformational changes between the GDP and GTP the association of the G protein with its GTP-specific complexes are located essentially in the switch I and partners, and it is usually very low (compiled in Zerial II regions as described for the related oncoprotein and Huber, 1995). The crystal structures of H-Ras define H-Ras. However, the mobile segments vary in length candidate residues for activation of the water molecule that and in the amplitude of movement. This suggests that attacks the γ-phosphate of GTP, and for the stabilization of even though similar regions might be involved in the the transition state of the GTPase reaction. It is not known, GDP-GTP cycle of small G proteins, the details of the however, how the GTPase reaction is designed to act as changes will be different for each G protein and will a timer and/or wait for interactions triggered by GTPaseensure the specificity of its interaction with a given set activating proteins (GAPs). of cellular proteins.Rap proteins, which include Rap1A, Rap1B, Rap2A Keywords: crystal structure/G proteins/GTP hydrolysis/ and Rap2B, have~50% sequence identity with Ras proteins Rap/Ras (reviewed in Bokoch, 1993). Rap1 was independently cloned by sequence homology (Pizon et al., 1988) and by its ability to revert the transformed ...

SSV1 is a virus infecting the extremely thermophilic archaeon Sulfolobus shibatae. The viral-encoded integrase is responsible for site-specific integration of SSV1 into its host genome. The recombinant enzyme was expressed in Escherichia coli, purified to homogeneity, and its biochemical properties investigated in vitro. We show that the SSV1 integrase belongs to the tyrosine recombinases family and that Tyr 314 is involved in the formation of a 3-phosphotyrosine intermediate. The integrase cleaves both strands of a synthetic substrate in a temperature-dependent reaction, the cleavage efficiency increasing with temperature. A discontinuity was observed in the Arrhenius plot above 50°C, suggesting that a conformational transition may occur in the integrase at this temperature. Analysis of cleavage time course suggested that noncovalent binding of the integrase to its substrate is rate-limiting in the cleavage reaction. The cleavage positions were localized on each side of the anticodon loop of the tRNA gene where SSV1 integration takes place. Finally, the SSV1 integrase is able to cut substrates harboring mismatches in the binding site. For the cleavage step, the chemical nature of the base in position ؊1 of cleavage seems to be more important than its pairing to the opposite strand.Site-specific recombination catalyzed by tyrosine recombinases plays a number of critical roles in prokaryote and eukaryote kingdoms. Well documented examples in lower eukaryotes and bacteria include generation of genetic variability, plasmid copy control and/or stable inheritance, resolution of bacterial chromosome dimers or viral DNA integration in host chromosomes (for reviews, see Refs. 1-3). Members of the tyrosine recombinases family catalyze site-specific recombination between two DNA sites by using a topoisomerase IB-like mechanism to cut and religate DNA strands (4, 5). Unlike topoisomerases, tyrosine recombinases perform the ligation step after strand exchange between the two DNA partners. Sitespecific recombination requires the assembly of a synaptic complex containing, at least, four enzyme protomers and the two DNA sites. The recombination reaction occurs by cutting and exchanging the two pairs of DNA strands in two temporally distinct steps. In the first step, cleavage occurs on the top strands of each DNA site. For each site, a 3Ј-phosphotyrosine DNA-protein covalent complex is formed and a free 5Ј-OH DNA end generated. The leaving strands then attack the phosphotyrosine link of the recombination partner, thus releasing the recombinase subunits. After this first round of strand cleavagestrand exchange, a Holliday junction is formed. The bottom strands are then cut and religated, thus resolving the Holliday junction and completing the recombination reaction.Site-specific recombination in archaea is not as well known. So far, the only studied system is SSV1, a virus of the extremely thermophilic archaeon Sulfolobus shibatae. In the cell, the 15.5-kb genome of SSV1 is present both as a circular DNA and as a provirus stably...