The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in bacterial virulence and infectivity. The P. aeruginosa genome contains three genes encoding acyl carrier proteins. We have cloned all three genes, expressed the acyl carrier proteins, and characterized each as a substrate for RhlI. A continuous, spectrophotometric assay was developed to facilitate kinetic and mechanistic studies of RhlI. Acp1, which has not been characterized previously, was a good substrate for RhlI, with a K(m) of 7 microM; the reaction proceeded with a k(cat) value of 0.35 s(-1). AcpP, which supports fatty acid biosynthesis, was also a good substrate in the RhlI reaction, where k(cat) was 0.46 s(-1), and the K(m) for AcpP was 6 microM. The third acyl carrier protein, Acp3, was a poor substrate for RhlI, with a K(m) of 280 microM; k(cat) was 0.03 s(-1). Taken together with microarray data from the literature which show that expression of the gene encoding Acp1 is under the control of the quorum-sensing system, our data suggest that Acp1 is likely to be the substrate for RhlI in vivo. Isotope labeling studies were conducted to investigate the chemical mechanism of the RhlI-catalyzed lactonization reaction. Solvent deuterons were not incorporated into product, which implicates a direct attack mechanism in which the carboxylate oxygen of the presumptive N-butyryl-SAM intermediate attacks the methylene carbon adjacent to the sulfonium ion. Alternative mechanisms, in which N-butyrylvinylglycine is formed via elimination of methylthioadenosine, were ruled out on the basis of the observation that RhlI failed to convert authentic N-butyrylvinylglycine to N-butyryl-L-homoserine lactone.

i-myo-inositol 1 -phosphate synthase (EC 5.5.1.4) from cyanobacteria1 (Spirulina platensis), alga1 (Euglena gracilis), and higher plant (Oryza sativa, Vigna radiata) sources was purified to electrophoretic homogeneity, biochemically characterized, and compared. Both chloroplastic and cytosolic forms of the enzyme were detected in E. gracilis, O. sativa, and V. radiata, whereas only the cytosolic form was detected in' streptomycin-bleached or chloroplastic mutants of E. gracilis and in S. platensis. Both the chloroplastic and cytosolic forms from different sources could be purified following the same three-step chromatographic protocol. i-myo-inositol 1 -phosphate synthases purified from these different sources do not differ significantly with respect to biochemical and kinetic parameters except for the molecular mass of the chloroplastic and cytosolic native holoenzymes, which appear to be homotetrameric and homotrimeric associations of their constituent subunits, respectively. Monovalent and divalent cations, sugar alcohols, and sugar phosphates are inhibitory to the enzyme activity. N-ethylmaleimide inhibition of synthase activity could be protected by the combined presence of the substrate glucose-6-phosphate and cofactor. NAD+. Antibody raised against the cytosolic enzyme from E. gracilis immunoprecipitates and cross-reacts with both chloroplastic and cytosolic forms from the other sources studied.Inositols are 6-C cyclohexane hexitols found ubiquitously in biological systems. Of the eight possible geometrica1 isomers, myo-inositol is the most abundant and occupies a central position in carbohydrate metabolism, being the precursor of a number of metabolic products such as inositol phosphates, phosphoinositides, cell wall polysaccharides, a number of pethylated derivatives, and IAA conjugates. As a free cyclitol, myo-inositol has been identified as a compound required for normal growth and development of fungal and plant tissue culture cells (Loewus and Dickinson, 1982;Loewus and Loewus, 1983;Biswas et al., 1984;Loewus, 1990). Depletion of the normal cellular level of inositol has been shown to lead to a loss of cell viability in the yeast Saccha-

The salt‐tolerant varieties of rice (Oryza sativa L.) exhibit enhanced activity of the chloroplast form of L‐myo‐inositol 1‐phosphate synthase (EC 5.5.4.1) under NaCl treatment either during the seedling stage or in fully grown plants during field growth. The salt‐induced enhancement was noticeable only in chloroplasts from light‐grown plants. The effects of these treatments on the cytosolic inositol synthase activity were less pronounced. While the effect of salt on the activity of the two forms was marginal in the salt‐sensitive varieties during seedling growth, salinity affected the chloroplast inositol synthase activity adversely in these varieties during growth of the plants under field conditions. The salt‐enhanced activities of inositol synthase(s) in the highly salt‐tolerant varieties studied were found to be comparable to that observed in Porteresia coarctata, a halophytic wild rice species. The implications of these findings, which suggest a role of the inositol pathway in osmoregulation, are discussed.

The gene encoding hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme, has been cloned from soybean (Glycine max). The gene encodes a protein that is 560 amino acids in length and contains a 31-amino acid signal sequence at the N terminus that is not present in the mature protein. The presence of two SKL motifs near the C terminus suggests that the protein resides in the peroxisome. This expectation is borne out by results from immunogold electron microscopy, which revealed that hydroxyisourate hydrolase was localized in the peroxisomes of uninfected root nodules. The gene encoding hydroxyisourate hydrolase was expressed in Escherichia coli, and soluble, catalytically active enzyme was purified to homogeneity. Sequence analysis revealed considerable homology with members of the ␤-glucosidase family of enzymes. Two glutamate residues, E199 and E408, align with the conserved glutamates that play catalytic roles in the ␤-glucosidases. However, the other residues that have been identified by crystallography to interact directly with the substrates in ␤-glucosidases are not conserved in hydroxyisourate hydrolase. The E199A and E408A hydroxyisourate hydrolase mutants were devoid of detectable catalytic activity. Analysis of transcripts for hydroxyisourate hydrolase demonstrated that its level of expression was highest in the nodule; mRNA was detectable 12 d after infection and increased until 21 d postinfection, then declined. In a similar manner, immunodetection of hydroxyisourate hydrolase indicated preferential localization in the nodule; the amount of protein detected was maximal at 21 d postinfection. The pattern of expression of hydroxyisourate hydrolase matched that of urate oxidase, and supports the hypothesis that hydroxyisourate hydrolase plays a role in ureide metabolism.Nitrogen is frequently the limiting nutrient for growth of many crop plants (Schubert, 1986), and tropical legumes such as soybean (Glycine max) obtain the nitrogen that ultimately supports protein synthesis via a particularly sophisticated system. The plants establish a symbiotic relationship with bacteria from the genus Bradyrhizobium, which contain the enzyme nitrogenase. Nitrogenase catalyzes the conversion of atmospheric dinitrogen to ammonia, which can be used to meet the plant's metabolic nitrogen requirements, but first it must be converted into organic forms that can be transported throughout the plant and further metabolized. Isotope-labeling studies demonstrate that ammonia produced from dinitrogen reduction is rapidly converted into allantoin and allantoate, the so-called ureides (Ohyama and Kumazawa, 1978); up to 95% of the nitrogen in the xylem sap in nodulated soybeans is in the form of ureides (Schubert, 1981).The ureides are derived from the oxidation of purines, and allantoate arises from the hydrolysis of S-allantoin, but details of the origin of allantoin remain unclear. It has been widely reported that allantoin is the product of the urate oxidase (UO) reaction, but recent work has established that UO catalyze...

The formation of N-butyrylhomoserine lactone catalyzed by RhlI has been investigated by transient-state kinetic methods. A single intermediate, assigned to N-butyryl- S-adenosylmethionine, was observed. Under single-turnover conditions, the intermediate formed with a rate constant of 4.0 +/- 0.2 s (-1) and decayed with a rate constant of 3.7 +/- 0.2 s (-1). No other intermediates were detected, demonstrating that the RhlI reaction proceeds via acylation of S-adenosylmethionine, followed by lactonization. S-Adenosylhomocysteine acted as a pseudosubstrate, in that it did not undergo either acylation or lactonization but did induce the deacylation of butyryl-acyl carrier protein. The K m for S-adenosylhomocysteine was approximately 15-fold higher than the K m for S-adenosylmethionine. The reactivities of acylated and unacylated sulfonium ions that were analogues of S-adenosylmethionine were investigated by computational methods. The calculations indicated that acylation of the substrate amino group activated the substrate for lactonization, by allowing the carboxyl group oxygen to approach more closely the methylene carbon to which it adds. This observation provides a satisfying chemical rationale for the order of the individual reactions in the catalytic cycle.