The involvement of nicotinamide adenine nucleotides (NAD ؉ , NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using 13 C and 31 P NMR to monitor in vivo the kinetics of the pools of NAD ؉ , NADH, ATP, inorganic phosphate (P i ), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of 13 C-labeled NAD ؉ and NADH. The capacity of L. lactis MG1363 to regenerate NAD ؉ was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH ؊ deficient strain was constructed by double crossover. Upon supply of glucose, NAD ؉ was constant and maximal (ϳ5 mM) in the parent strain (MG1363) but decreased abruptly in the LDH ؊ strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH ؊ strain and NADH reached high levels (2.5 mM), reflecting severe limitation in regenerating NAD ؉ . However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mM. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P i content could play an important role.Lactococcus lactis plays an essential role in the manufacture of a wide range of dairy products. The relative simplicity of L. lactis metabolism that converts sugars via the glycolytic pathway to pyruvate, generating energy mainly through substrate level phosphorylation, makes it an attractive model organism to test metabolic engineering strategies. Moreover, the large number of genetic tools available for L. lactis (1) and the recent release of the complete genome sequence are additional incentives to study the physiology of this organism in great depth (2).Despite numerous studies, a satisfactory answer to the question, What controls the glycolytic flux in L. lactis? has not been put forward. During homolactic fermentation, regulation of the carbon flux has been associated with high levels of fructose 1,6-bisphosphate (FBP), 1 which activates lactate dehydrogenase (LDH; EC 1.1.1.27) and pyruvate kinase (PK; EC 2.7.1.40), directing the flux toward the production of lactate (3). A metabolic shift from homolactic (lactate production) to mixed acid fermentation (ethanol, acetate, and formate production) was observed in glucose-limited chemostat cultures (4). A deviation from homolactic fermentation was also reported under aerobic conditions (5) or during the metabolism of galactose (6). The format...

Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.One of the most striking characteristics of extremophiles is their ability to thrive under environmental conditions that would be lethal to most organisms. In particular, hyperthermophiles, having optimal growth temperatures above 80°C (4), pose intriguing questions regarding the biochemical strategies used for the adaptation of their cellular structures to withstand such high temperatures. Maintaining protein performance at high temperature could be accomplished by a number of mechanisms: (i) intrinsic thermostability, (ii) increased turnover, (iii) improved action of molecular chaperones, and (iv) extrinsic stabilization by compatible solutes (13). Although most enzymes from thermophilic sources show an intrinsic thermostability higher than that of their mesophilic counterparts, several enzymes derived from hyperthermophilic sources show an unexpectedly low intrinsic stability in vitro (13,14). Therefore, extrinsic factors, such as compatible solutes, may play a role in the thermostabilization of these cellular components.Some compatible solutes, namely, glutamate, betaine, and trehalose, are widespread in mesophilic organisms, but compatible solutes unique to thermophiles and hyperthermophiles have also been identified in recent years (8; H. Santos and M. S. da Costa, submitted for publication). Newly discovered solutes from thermophilic and hyperthermophilic organisms include cyclic-2,3-bisphosphoglycerate (17), two isomers of dimyo-inositol phosphate (25, 31), mannosylglycerate and mannosylglyceramide (24, 28, 36), di-mannosyl-di-myo-inosito...

The environment-dependent multiexponential behavior of N-acetyltyrosinamide (NAYA) and other tyrosine derivatives is revisited, aiming for a better understanding of tyrosine as an intrinsic fluorescent probe for protein microenvironment changes during conformational changes. The effects of solvent polarity, viscosity, and temperature on the fluorescence decay of NAYA were evaluated using dioxane-water mixtures and pure solvents. Double-exponential decays were observed in dioxane-water mixtures above 70% (v/v) water concentration including pure water, for temperatures below 50 °C. However, at higher temperatures, or in dioxane-water mixtures with lower water concentrations, NAYA shows single-exponential decays. Singleexponential decays were also generally observed in pure solvents (dioxane, acetonitrile, methanol, ethanol, DMSO). The exception was the strong hydrogen-bond donor trifluorethanol, in which NAYA decays as a double exponential. The results are consistent with a solvent-modulated excited-state intramolecular electron transfer from the phenol to the amide moiety occurring in one of the three rotamers of NAYA. On the basis of a full kinetic analysis of the data, it is shown that experimental observation of double-exponential decays depends on three factors: the ground-state population of rotamers, their rates of interconversion (k r ) 4.4 × 10 8 s -1 and k r ′ ) 5.2 × 10 8 s -1 , in water at 23 °C, E r ) 4.9 ( 0.1 kcal mol -1 and E r ′) 5.2 ( 0.3 kcal mol -1 ), and the electron-transfer rate constant (k ET ) 2.0 × 10 9 s -1 , in water at 23 °C, E ET ) 1.8 ( 0.2 kcal mol -1 ). Solvent viscosity controls the interconversion rate constants while solvent polarity and hydrogen-bonding ability determines the Gibbs energy of electron transfer and the magnitude of its rate constant. Consequently, the nature of tyrosine decays in proteins is determined from a delicate balance between the interconversion and electron-transfer rate constants.