Dale B. Karr

Improved two-step method for the assay of adenylate and guanylate cyclase

Abstract A two-step assay for adenylate and guanylate cyclase is described utilizing α- 32 P-labeled ATP or GTP as substrate and involving purification of the resulting 32 P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 m m HCl for cGMP and 10 m m HClO 4 for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the 32 P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.

Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms

Internally seedborne microorganisms are those surviving common surface sterilization procedures. Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum. Bacterial isolates from surface-sterilized soybean seed, cv. Williams 82 and cv. Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis. Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G. The effects of this antibiotic on seedling development and on B. japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here. Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development. No differences in nodulation kinetics, nitrogen fixation onset or rates were observed. However, the number of B. japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment. Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B. japonicum to the soybean root, without affecting plant development.

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min–1 (mg protein)–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent Km of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the Vmax nor the d,l-isocitrate Km of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum.

Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

Background: UDP-galactopyranose mutase (UGM) catalyzes a step in galactofuranose biosynthesis in pathogens and is a promising drug design target. Results: The first crystal structures and SAXS analysis of UGM from the pathogenic fungus Aspergillus fumigatus are reported. Conclusion: The unique quaternary structure enables profound conformational changes to occur upon substrate binding. The structures support the covalent mechanism. Significance: The structures should aid inhibitor design. UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 Å to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation.

Abstract. The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation.

Breaking the covalent connection: Chain connectivity and the catalytic reaction of PMM/PGM

Fragment complementation has been used to investigate the role of chain connectivity in the catalytic reaction of phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, a human pathogen. A heterodimer of PMM/PGM, created from fragments corresponding to its first three and fourth domains, was constructed and enzyme activity reconstituted. NMR spectra demonstrate that the fragment corresponding to the fourth (C‐terminal) domain exists as a highly structured, independent folding domain, consistent with its varied conformation observed in enzyme–substrate complexes. Steady‐state kinetics and thermodynamics studies reported here show that complete conformational freedom of Domain 4, because of the break in the polypeptide chain, is deleterious to catalytic efficiency primarily as a consequence of increased entropy. This extends observations from studies of the intact enzyme, which showed that the degree of flexibility of a hinge region is controlled by the precise sequence of amino acids optimized through evolutionary constraints. This work also sheds light on the functional advantage gained by combining separate folding domains into a single polypeptide chain.

Further evidence for the uniformity of the microsymbiont population from soybean nodules.

Summary The microsymbiont population in soybean root nodules (Glycine max L.cv.Williams 82 inoculated with Bradyrhizobium japonicum 2143) was characterized during symbiotic development.The microsymbiont population obtained from nodules of plants at different ages was subjected to sucrose density centrifugation.The microsymbionts were fractionated according to their buoyant densities and characterized with regard to several enzyme activities and O2 uptake.No differences were observed in the specific activities of these enzymes in the microsymbionts obtained at various buoyant densities.This would suggest the absence of discrete subpopulations.These results are consistent with the concept of a single microsymbiont population.

Three crystal forms of the bifunctional enzyme proline utilization A (PutA) from Bradyrhizobium japonicum

Proline utilization A proteins (PutAs) are large (1000-1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and Delta(1)-pyrroline-5-carboxylate dehydrogenase domains. Here, the first successful crystallization efforts for a PutA protein are described. Three crystal forms of PutA from Bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic. The apparent tetragonal and hexagonal crystals were grown in the presence of PEG 3350 and sodium formate near pH 7. The apparent tetragonal form diffracted to 2.7 A resolution and exhibited pseudo-merohedral twinning such that the true space group is P2(1)2(1)2(1) with four molecules in the asymmetric unit. The hexagonal form diffracted to 2.3 A resolution and belonged to space group P6(2)22 with one molecule in the asymmetric unit. Centered monoclinic crystals were grown in ammonium sulfate, diffracted to 2.3 A resolution and had two molecules in the asymmetric unit. Removing the histidine tag was important in order to obtain the C2 crystal form.

Purification of an Autophosphorylating Protein from Imbibing Soybean (Glycine max L.) Seed Exudate and its Identification as a Nucleoside Diphosphate Kinase

Summary A calcium-independent autophosphorylating protein was identified in the exudate of imbibed soybean ( Glycine max L.) seeds. The inclusion of histone I or calmodulin in protein phosphorylation assays had no observable affect on the extent of phosphorylation of this protein. The protein was purified to apparent homogeneity, had a native molecular mass of 72 Ku, a subunit molecular mass of 18 Ku, and was capable of in situ phosphorylation. The phosphorylated residue was identified as serine by two-dimensional TLE analysis. Polyclonal antibodies were raised against the purified protein and were used to isolate a eDNA clone from a lambda-ZAP soybean eDNA expression library. The nucleotide sequence of the clone and its deduced amino acid sequence had very high homology to those of nucleoside diphosphate kinases (NDP kinase, EC 2.7.4.6) from plants and other eukaryotic tissue. In addition, the purified autophosphorylating protein exhibited NDP kinase activity. The purified enzyme preferentially transferred the terminal phosphate from ATP to a purine, but no transfer to the pyrimidine, CDP, was observed.

Recovery of nitrogenase from aerobically isolated soybean nodule bacteroids

Bacteroids, the symbiotic forms of rhizobia, express nitrogenase, which is the enzyme that catalyzes the reduction of atmospheric dinitrogen to ammonium. The extreme oxygen lability of nitrogenase requires that bacteroids be isolated under anaerobic conditions to preserve their ability to reduce atmospheric dinitrogen. Aerobically isolated bacteroids were found to exhibit nitrogenase activity as measured via the acetylene reduction method but only after incubation of the bacteroids for about 1 h under low partial pressures of oxygen. The recovery of nitrogenase activity was dependent upon the partial pressure of oxygen to which the bacteroids were exposed during the one-hour incubation. The recovery of acetylene reduction activity was prevented by addition of inhibitors of protein synthesis, but not by inhibitors of transcription. Acetylene reduction could be expressed from aerobically isolated bacteroids stored for up to three days after isolation. The recovery of nitrogenase demonstrates that both the plant and the bacteroids possess mechanisms to protect the enzyme against oxygen. The recovery of nitrogen fixation activity from aerobically isolated bacteroids may provide a facile way to obtain bacteroids for studies on symbiotic functioning.