31P- and 13C-NMR studies on the flavin-protein and flavin-ligand interactions in brewer's yeast old yellow enzyme

The 31P- and 13C-NMR spectra of old yellow enzyme (OYE) were measured. The 31P-NMR signal of FMN bound to apo OYE-I, one of the pure forms of OYE, was observed at a substantially lower field compared to that of free FMN. While the 31P-signal of free FMN is pH-titratable with a pK value of about 6.5, which corresponds to the monoanion-dianion transition of the phosphate group, the 31P-signal of FMN bound to OYE-I shows no pH-dependence at pH 5-9, indicating that the phosphate group of FMN bound to OYE-I is fixed in the dianionic form in the pH region of 5-9. Apo OYE(0), i.e., the OYE preparation obtained by the conventional method, was reconstituted with [2-13C]FMN or [4,10a-13C2]FMN, while apo OYE-I was reconstituted with [4a-13C]FMN. The 13C-NMR spectra of these reconstituted OYE species were measured in the absence and presence of phenolic compounds which form complexes with OYE. Each 13C-signal of the 13C-labeled FMN became broader in the bound state compared to the free state, indicating restriction of flavin mobility in the bound form. Complex formation of the reconstituted OYE species with p-bromophenol did not shift the 10a-13C signal but shifted the 2- and 4-13C signals slightly upfield, whereas the 4a-13C signal was shifted significantly upfield in the complexed form. This complex-induced upfield shift of the 4a-13C signal was measured with various p-substituted phenols.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of glutamine 114 in old yellow enzyme

Glutamine 114 of OYE1 is a well conserved residue in the active site of the Old Yellow Enzyme family. It forms hydrogen bonds to the O2 and N3 of the flavoprotein prosthetic group, FMN. Glutamine 114 was mutated to asparagine, introducing an R-group that is one methylene group shorter. The resultant enzyme was characterized to determine the effect of the mutation on the mechanistic behavior of the enzyme, and the crystal structure was solved to determine the effect of the mutation on the structure of the protein. The Q114N mutation results in little change in the protein structure, moving the amide group of residue 114 out of H-bonding distance, allowing repositioning of the FMN prosthetic group to form new interactions that replace the lost H-bonds. The mutation decreases the ability to bind ligands, as all dissociation constants for substituted phenols are larger than for the wild type enzyme. The rate constant for the reductive half-reaction with beta-NADPH is slightly greater, whereas that for the oxidative half-reaction with 2-cyclohexenone is smaller than for the wild type enzyme. Oxidation with molecular oxygen is biphasic and involves formation and reaction with O(2), a phenomenon that is more pronounced with this mutation than with wild type enzyme. When superoxide dismutase is added to the reaction, we observe a single-phase reaction typical of the wild type enzyme. Turnover reactions using beta-NADPH with 2-cyclohexenone and molecular oxygen were studied to further characterize the mutant enzyme.     

The nucleotide sequence of asparagine tRNA from brewer's yeast

The nucleotide sequence of asparagine tRNA from brewer's yeast has been determined using postlabeling methods. The primary structure is as follows: pG-A-C-U-C-C-A-U-G-m2G-C-C-A-A-G-D-D-G-G-D-D-A-A-G-G-C-m2 2G- U-G-C-G-A-C-U-G-U-U -t6A-A-psi-C-G-C-A-A-G-A-D-m5C-G-U-G-A-G-T-psi-C-A-m1A-C-C-C-U-C-A-C-U-G -G-G-G- U -C-G-C-C-A. Its anticodon G-U-U can recognize the two codons for asparagine.     

Wort sugar uptake by brewer's yeast

Summary A culture of brewer's yeast,Saccharomyces cerevisiae (NCYC 240), maintained on Wickerham's MYGP medium, utilized the principal wort sugars sequentially in the order glucose-maltose-maltotriose, when inoculated into brewer's wort. A culture of the same strain maintained on brewer's wort utilized these three sugars simultaneously. Simultaneous utilization could be induced in MYGP-maintained cultures by successive sub-culture in brewer's wort, and appears to be the general rule of sugar uptake during wort fermentation under brewery conditions.     

Biotransformation of explosives by the old yellow enzyme family of flavoproteins

Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.     

Flocculation of cell walls of brewer's yeast and effects of metal ions,protein-denaturants and enzyme treatments

Effects of metal ions, protein-denaturants and enzyme treatments on flocculation of cell walls of Beer Yeast IFO 2018 were investigated. Cell walls from flocculent cells grown in a complete medium were able to form flocs as were whole cells, but cell walls from non-flocculent cells, such as “Mg²⁺-deficient” cells, “early-phase” cells and “low-pH” cells, were not. The cell walls dispersed in distilled water reflocculated in solutions containing Ca²⁺ or other metal ions. Of the alkali metal ions tested, only Na⁺ inhibited flocculation of flocculent cell walls at a concentration more than 0.1 M. Ca²⁺ or Sn⁴⁺ was absolutely required for flocculation of cell walls in the physiological saline (NaCl, 150 mM), but the effect of Sn⁴⁺ seems rather non-specific, because it promoted flocculation of non-flocculent cell walls as well. Sr²⁺ and Ba²⁺ were antagonistic to Ca²⁺ and inhibited flocculation. Flocculation of cell walls was also depressed by high concentrations of protein-denaturants, e.g. urea and guanidine·HCl. Treatment with proteolytic enzymes deprived cell walls of floc-forming ability. Effect of metal ions, protein-denaturants and treatment with enzymes on the flocculation of intact cells was investigated as control. Since flocculating properties of cell walls were very similar to those of intact cells, flocculation must be an inherent property of cell walls.     

Insulin-like activity of chromium-binding fractions from brewer's yeast

51CrCl3 was added to the incubation medium of Saccharomyces cerevisiae for up to 48 hr. After repeated freezing and thawing, lysing in 9 M urea with 1% NP-40 detergent, and dialysis against water, the lower molecular weight (Mr less than 3500) dialysate was retained on a SE53 cationic exchange column, eluted with 0.25 M NH4OH and fractionated on a Bio-gel P-2 column. The insulin-like biological activity of the fractions was measured by the 14C-glucose oxidation in isolated rat adipocytes. The biological activity that was found in two of nine fractions did not correspond to their chromium content. Moreover, identical findings were obtained when chromium was added not to the live yeast but to the yeast extract, which showed that its binding was a chemical process not requiring cellular activity. No fraction demonstrated insulin-potentiating activity on rat adipocytes.     

Enhanced transformation of TNT by Arabidopsis plants expressing an old yellow enzyme

2,4,6-Trinitrotoluene (TNT) is released in nature from manufacturing or demilitarization facilities, as well as after the firing or detonation of munitions or leakage from explosive remnants of war. Environmental contamination by TNT is associated with human health risks, necessitating the development of cost-effective remediation techniques. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. In this study, we present a system for TNT phytoremediation by overexpressing the old yellow enzyme (OYE3) gene from Saccharomyces cerevisiae. The resulting transgenic Arabidopsis plants demonstrated significantly enhanced TNT tolerances and a strikingly higher capacity to remove TNT from their media. The current work indicates that S. cerevisiae OYE3 overexpression in Arabidopsis is an efficient method for the phytoremoval and degradation of TNT. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TNT.     

Reactivity of old yellow enzyme with alpha-NADPH and other pyridine nucleotide derivatives

The reaction of Old Yellow Enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. alpha-NADPH, beta-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 microM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both alpha- and beta-NADPH are capable of reducing OYE, and alpha-NADPH is more effective. Using beta-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with alpha-NADPH was significantly higher than that with beta-NADPH.