The thiobarbituric acid reacting material produced during enzymatic microsomal lipid peroxidation has been identified as malonaldehyde. The malonaldehyde was condensed with urea to form 2‐hydroxy‐pyrimidine, which was identified by its ultraviolet spectrum, chromato‐graphic properties, and mass spectrum. Incubations with phosphatidyl choline labelled with tritiated arachidonate yielded 2‐hydroxy‐pyrimidine with a specific activity nearly equal to that of the phospholipid arachidonate. Incubations with tritiated arachidonic acid yielded 2‐hydroxy‐pyrimidine with a specific activity nearly 2 orders of magnitude less than that of free arachidonic acid. Therefore, phospholipid arachidonate has been established as the major source of the malonaldehyde produced during microsomal lipid peroxidation.

In Escherichia coli, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was dimeric, dependent on Mn 2؉ for activity, and exhibited an apparent K m of 35 M for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.Growth of Escherichia coli on glycerol or sn-glycerol 3-phosphate (glycerol-P) as the sole carbon source is mediated primarily by the glp regulon (15). The glpFKX operon, one of the five operons of the regulon, encodes glycerol facilitator (glpF), glycerol kinase (glpK), and a protein of unknown function (glpX) (31, 32). It was initially reported that GlpX displays limited sequence similarity to the Synechococcus leopoliensis fructose 1,6-bisphosphatase (FBPase) (31). In our work, a more recent BLAST search revealed that GlpX manifests 39% identity to an FBPase of Synechococcus sp. strain PCC7492 (29). Until now, the only recognized E. coli FBPase was encoded by fbp (25). This FBPase (FBPase I) has only 10% identity to the amino acid sequence of glpX-encoded FBPase (FBPase II). E. coli FBPase I is dependent on Mg 2ϩ , is inhibited by low levels of AMP, is tetrameric (1), and is necessary for growth of E. coli on gluconeogenic substrates such as glycerol or succinate (10).It is not clear why E. coli would maintain two distinct FBPases. FBPases can modulate the concentration of fructose 1,6-bisphosphate [Fru(1,6)P 2 ] and fructose 6-phosphate. These two regulatory hexoses affect glycolysis enzymes 6-phosphofructokinases I and II, pyruvate kinase I, and phosphoenolpyruvate (PEP) carboxylase (2,4,13,20); glycogen synthesis enzyme ADP-glucose pyrophosphorylase (12); and carbonsource import pathway enzymes glycerol kinase and 1-phosphofructokinase (6, 15). Flux through the Embden-Meyerhof pathway in the direction of glycolysis or gluconeogenesis can be allosterically controlled at the enzyme level by other metabolites as well: PEP, ATP, ADP or AMP (9). The potential "futile cycle" of phosphofructokinases and FBPases is also alleviated by this regulation. Therefore, regulation of FBPases is important.In this communication, the FBPase activity of the glpX gene product is documented. The glpX-encoded enzyme, FBPase II, was purified and characterized, enabling comparison of the attributes of E. coli FBPases in vitro. Further, a chromosomal insertion mutation in glpX was constructed to test the physiological effects of the glpX mutation on carbohydrate metabolism.E. coli strains and cloning of glpX. E. coli strains used in this study are listed in Table 1. Strains were grown in Luria broth (LB) supplemented with antibiotics as needed or in minimal medium (7) containing 0.4% glycerol or 0.2% glucose.The glpX gene was PCR amplified from chromosomal DNA of strain MG1655 using the primer pair acgtgaaTTCCCCTG TGCT...

Nitrogenase catalyzes the biological reduction of N 2 to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HCϵCH) to ethylene (H 2 C‫؍‬CH 2 ). It is known that the metallocluster FeMo-cofactor located within the nitrogenase MoFe protein component provides the site of substrate reduction, but the exact site where substrates bind and are reduced on the FeMo-cofactor remains unknown. We have recently shown that the ␣-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; ␣-70 approaches one face of the FeMo-cofactor, and when valine is substituted by alanine at this position, the substituted nitrogenase is able to accommodate a reduction of the larger alkyne propargyl alcohol (HCϵCCH 2 OH, propargyl-OH). During this reduction, a substrate-derived intermediate can be trapped on the FeMo-cofactor resulting in an S ‫؍‬ 1/2 spin system with a novel electron paramagnetic resonance spectrum. In the present work, trapping of the propargyl-OH-derived or propargyl amine (HCϵCCH 2 NH 2 , propargyl-NH 2 )-derived intermediates is shown to be dependent on pH and the presence of histidine at position ␣-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the -OH group or the -NH 3 ؉ group and the imidazole ⑀-NH of ␣-195 His . Thus, for the first time it is possible to establish the location of a bound substrate-derived intermediate on the FeMo-cofactor. Refinement of the binding mode and site was accomplished by the use of density functional and force field calculations pointing to an 2 coordination at Fe-6 of the FeMo-cofactor.Nitrogenase is comprised of two component proteins, called the iron protein and the MoFe protein, which together catalyze the nucleotide-dependent reduction of N 2 to ammonia (Equation 1). N 2 ϩ 8e Ϫ ϩ 16MgATP ϩ 8H ϩ 3 2NH 3 ϩ H 2 ϩ 16MgADP ϩ 16P i (Eq. 1) During catalysis, electrons are delivered one at a time from the iron protein to the MoFe protein in a reaction coupled to the hydrolysis of 2 eq of MgATP for each equivalent of electrons transferred (1, 2). The MoFe protein contains two metalloclusters called the P-cluster [8Fe-7S] and the FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], where X is proposed to be nitrogen, carbon, or oxygen (3). The P-clusters are thought to mediate electron transfer from the iron protein to the FeMo-cofactor, which in turn provides the site for substrate binding and reduction. The structure of the FeMo-cofactor has been elucidated from the solution of x-ray structures of MoFe proteins (3-7), yet where and how substrates interact with the FeMocofactor is still unknown. Different models for where substrates bind to the FeMo-cofactor have been developed; they were built on evidence from model compounds, theoretical calculations, and kinetic and biophysical studies on the wildtype (WT) 1 and genetically altered MoFe proteins (8). Some models propose binding and reduction of substrates at th...

Cryptococcus neoformans produces large amounts of the acyclic hexitol mannitol in culture and infected animals, but the functional and pathogenic significance of mannitol production by this fungus is not known. We exposed C. neoformans H99 (Cn H99) to UV irradiation (1 x LD, , ) and screened survivors for mannitol production. A mutant, Cn MLP (Mannitol Low Producer), synthesized less mannitol from glucose (2.7 vs 8-2 nmol per lo8 cells min-' a t 37 "C) and contained less intracellular mannitol (1 vs 11 pmol per lo6 cells a t 37 "C) than did Cn H99. Cn MLP and Cn H99 were similar with respect to carbon assimilation patterns, rates of glucose consumption, growth rates a t 30 "C, urease and phenoloxidase activities, morphology, capsule formation, mating type, electrophoretic karyotype, rapid amplification of polymorphic DNA (RAPD) patterns and antifungal susceptibility. However, Cn MLP was more susceptible than was Cn H99 to growth inhibition and killing by heat and high NaCl concentrations. Also, the LD, , values in mice injected intravenously were 3.7 x lo6 c.f.u. for Cn MLP compared to 6 9 x lo2 c.f.u. for Cn H99. Moreover, 500 c.f.u. Cn H99 intravenously killed 12 of 12 mice by 60 d, whereas all mice given the same inoculum of Cn MLP survived. Classical genetic studies were undertaken to determine if these differences were due to a single mutation, but the basidiospores were nonviable. These results suggest that the abilities of C. neoformans to produce and accumulate mannitol may influence its tolerance to heat and osmotic stresses and its pathogenicity in mice.

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