Change in Cofactor Specificity of Oxidoreductases by Adaptive Evolution of an Escherichia coli NADPH-Auxotrophic Strain

In the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism.

Substitution of a Surface-Exposed Residue Involved in an Allosteric Network Enhances Tryptophan Synthase Function in Cells

Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (βTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. We show here that substitution of a distant, surface-exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating βTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan-auxotrophic strain of Escherichia coli compared to complementation with wild-type αTS, emphasizing the biological importance of the network. Surface-exposed networks provide new opportunities in allosteric drug design and protein engineering, and hint at potential information conduits through which the functions of a metabolon or even larger proteome might be coordinated and regulated.

A role of Candida albicans CDC4 in the negative regulation of biofilm formation

The CDC4 gene is nonessential in Candida albicans and plays a role in suppressing filamentous growth, in contrast to its homologues, which are involved in the G1–S transition of the cell cycle. While characterizing the function of C. albicans CDC4 (CaCDC4), we found that the loss of CaCDC4 resulted in a reduction in cell flocculation, indicating a possible role for CaCDC4 in biofilm formation. To elucidate the role of CaCDC4 in biofilm formation, Cacdc4 null mutant strains were constructed by using the mini-Ura-blaster method. To create a CaCDC4 rescued strain, the plasmid p6HF-ACT1p-CaCDC4 capable of constitutively expressing CaCDC4 was introduced into the Cacdc4 homozygous null mutant. To determine the biofilm formation ability, an in vitro XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-5-carboxanilide) reduction assay was used. Compared with the parental auxotrophic strain BWP17, the Cacdc4 homozygous null mutant was able to enhance biofilm formation significantly. This enhancement of biofilm formation in the Cacdc4 homozygous null mutant could be reversed by constitutively expressing CaCDC4. We conclude that CaCDC4 has a role in suppressing biofilm formation in C. albicans.

Pantothenate and Pantetheine Antagonize the Antitubercular Activity of Pyrazinamide

ABSTRACTPyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth ofMycobacterium tuberculosisvia an as yet undefined mechanism. AnM. tuberculosislaboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains ofM. tuberculosis. It was found that pantothenate and other β-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that β-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, includingn-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates withpanD-linked resistance.

Saccharomyces cerevisiae Multidrug Resistance Transporter Qdr2 Is Implicated in Potassium Uptake, Providing a Physiological Advantage to Quinidine-Stressed Cells

ABSTRACT The QDR2 gene of Saccharomyces cerevisiae encodes a putative plasma membrane drug:H+ antiporter that confers resistance against quinidine, barban, bleomycin, and cisplatin. This work provides experimental evidence of defective K+ (Rb+) uptake in the absence of QDR2. The direct involvement of Qdr2p in K+ uptake is reinforced by the fact that increased K+ (Rb+) uptake due to QDR2 expression is independent of the Trk1p/Trk2p system. QDR2 expression confers a physiological advantage for the yeast cell during the onset of K+ limited growth, due either to a limiting level of K+ in the growth medium or to the presence of quinidine. This drug decreases the K+ uptake rate and K+ accumulation in the yeast cell, especially in the Δqdr2 mutant. Qdr2p also helps to sustain the decrease of intracellular pH in quinidine-stressed cells in growth medium at pH 5.5 by indirectly promoting H+ extrusion affected by the drug. The results are consistent with the hypothesis that Qdr2p may also couple K+ movement with substrate export, presumably with quinidine. Other clues to the biological role of QDR2 in the yeast cell come from two additional lines of experimental evidence. First, QDR2 transcription is activated under nitrogen (NH4 +) limitation or when the auxotrophic strain examined enters stationary phase due to leucine limitation, this regulation being dependent on general amino acid control by Gcn4p. Second, the amino acid pool is higher in Δqdr2 cells than in wild-type cells, indicating that QDR2 expression is, directly or indirectly, involved in amino acid homeostasis.