Barrie Entsch

Monitoring changing toxigenicity of a cyanobacterial bloom by molecular methods.

ABSTRACT Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the β and α subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.

Regulators of cell division in plant tissues

The cytokinins in certain fractions prepared from extracts of immature sweet-corn (Zea mays L.) kernels using polystyrene ion-exchange resins have been further investigated. Cytokinins active in the radish cotyledon bioassay were purified from these fractions and identified as 9-β-D-glucopyranosylzeatin, 9-β-D-glucopyranosyldihydrozeatin, O-β-D-glucopyranosylzeatin. and O-β-D-glucopyranosyl-9-β-D-ribofuranosylzeatin. In addition, compounds which resemble zeatin and its glycosides in chromatographic behaviour and in ultraviolet absorption characteristics were purified from extracts of the same material by high-performance liquid chromatography. In addition to zeatin and zeatin riboside, the following compounds were identified unambiguously: O-β-D-glucopyranosyl-9-β-D-ribofuranosyldihydrozeatin, O-β-D-glucopyranosyldihydrozeatin, and hihydrozeatin riboside. A further compound was tentatively identified as O-β-D-glucopyranosylzeatin, and at least two unidentified compounds appeared to be new derivatives of zeatin. In identifying the above compounds, chemical-ionization mass spectrometry proved to be an invaluable complementary technique, yielding spectra showing intense protonated-molecular-ion peaks and also prominent structure-related fragmentation that was either not evident or very minor in the electron-impact spectra. An assessment of the relative importance of the various possible mechanisms for cytokinin modification and inactivation in mature sweet-corn kernels was made by supplying [3H]zeatin and [3H]zeatin riboside to such kernels after excision. The principal metabolites of zeatin were adenine nucleotides, adenosine and adenine, while little of the metabolite radioactivity was attributable to known O-glucosides. Adenine nucleotides and adenine were the principal metabolites of zeatin riboside, while lesser metabolites were identified as adenosine, dihydrozeatin, and the O-glucosides of dihydrozeatin and dihydrozeatin riboside. Side-chain cleavage, rather than side-chain modification, appears to be the dominant form of cytokinin metabolism in mature sweet-corn kernels.

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large βαβ domain (residues 1–180) and the smaller sheet domain (residues 180–270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.

Inhibitors of two enzymes which metabolize cytokinins

Abstract Compounds which inhibit the natural metabolic inactivation of cytokinins are of considerable physiological significance. In this study, inhibitors have been found for two enzymes which form glucose and alanine conjugates of cytokinin bases, namely, cytokinin 7-glucosyltransferase and β-(9-cytokinin)alanine synthase. The most effective inhibitors found for the former enzyme were the cytokinin analogues 3-methyl-7-n-pentylaminopyrazolo[4,3-d]pyrimidine, which acted competitively (Ki, 22 μM), and the diaminopurine, 6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine (Ki, 3.3 μM). However these compounds were ineffective as inhibitors of the cytokinin-alanine synthase which was inhibited competitively by IAA (Ki 70 μM) and related compounds, especially 5,7-dichloro-IAA (Ki 0.4 μM). Certain urea derivatives were moderately effective inhibitors of the enzymes (Kica 100μM).

Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii *

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). The enzyme system is composed of two proteins: an FMN reductase (C1) and an oxygenase that uses FMNH– (C2). We report detailed transient kinetics studies at 4 °C of the reaction mechanism of C2.C2 binds rapidly and tightly to reduced FMN (Kd, 1.2 ± 0.2 μm), but less tightly to oxidized FMN (Kd, 250 ± 50 μm). The complex of C -FMNH–2 reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 ± 0.1 × 106 m–1 s–1, whereas the C -FMNH–2 -HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k = 4.8 ± 0.2 × 104 m–1 s–1). The kinetic mechanism of C2 was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C -FMNH–2 complex, but the preferred path was oxygen reacting with C -FMNH–2 to form the C(4a)-hydroperoxy-FMN intermediate prior to HPA binding. Hydroxylation occurs from the ternary complex with a rate constant of 20 s–1 to form the C2-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (>0.5 mm), HPA formed a dead end complex with the C2-C(4a)-hydroxy-FMN intermediate (similar to single component flavoprotein hydroxylases), thus inhibiting the bound flavin from returning to the oxidized form. When FADH– was used, C(4a)-hydroperoxy-FAD, C(4a)-hydroxy-FAD, and product were formed at rates similar to those with FMNH–. Thus, C2 has the unusual ability to use both common flavin cofactors in catalysis.

Preparation and characterization, using high-performance liquid chromatography, of an enzyme forming glucosides of cytokinins.

Cytokinins can occur naturally as glycosides with beta-D-glucose as the sugar substituent. From radish (Raphanus sativus) cotyledons, an enzyme has been partly purified which synthesizes the 7-glucopyranoside of zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine], a compound known to occur in this species. High-performance reverse-phase liquid chromatography was uniquely useful as the analytical procedure for quantitative study of the minute amounts of enzyme available. The enzyme uses UDPglucose as the source of the sugar residue. A large number of derivatives of purine are glucosylated, but adenine derivatives with an alkyl side chain at least three carbon atoms in length at position N6 are preferentially glucosylated. This corresponds to the structural features required for high cytokinin activity. The 7-glucoside of zeatin is known to be very weakly active in cytokinin bioassays. Hence, this enzyme, and others catalyzing the same reaction, have a role in the regulation of cytokinin activity.

Sequence and organization of pobA, the gene coding for p-hydroxybenzoate hydroxylase, an inducible enzyme from Pseudomonas aeruginosa

The only recognized gene for the metabolism of p-hydroxybenzoate in Pseudomonads (pobA) has been isolated from Pseudomonas aeruginosa to provide the DNA for mutagenesis studies of the protein product, p-hydroxybenzoate hydroxylase. Since pobA is induced by p-hydroxybenzoate to produce large amounts of enzyme, its regulation in P. aeruginosa is significant. The nucleotide sequence of pobA is presented with the derived amino acid (aa) sequence, which has only two substitutions compared to the amino acid sequence obtained from the enzyme from P. fluorescens. The derived amino acid sequence predicts that the enzyme is a single polypeptide of 394 aa residues and contains one molecule of FAD. The complete structure of the protein from P. aeruginosa can be derived by analogy from the published structure of the protein from P. fluorescens. Transcription mapping was used to determine that there is one site for the initiation of mRNA synthesis in P. aeruginosa. The presence of a putative operator in the sequence suggests primary regulation by a repressor protein which binds p-hydroxybenzoate. The ribosome-binding site permits translation of the gene in Escherichia coli at levels comparable to its production in P. aeruginosa, but it produces no detectable product in E. coli under the influence of its own promoter sequence. The promoter does not conform to the common consensus sequence of E. coli promoters. The results have identified an apparent novel promoter for P. aeruginosa, which may reflect the presence of a sigma factor required for pobA induction. Repression of expression by glucose suggests a binding site in the sequence for catabolite repression.

LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence

The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4 degrees C pH 8.0, Km for NADH, Km for FMN, and kcat were calculated to be 15.1 microM, 2.7 microM, and 1.7 s(-1), respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH- for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH- for the luminescence reaction in vivo.

Enzymic glucosylation of the cytokinin, 6-benzylaminopurine

Abstract Two separate enzyme activities for the formation of the 7- and 9-glucopyranosyl derivatives of the cytokinin 6-benzylaminopurine (BAP), have been found in soluble extracts of expanded cotyledons of radish ( Raphanus sativus ). The enzymes have been purified partially by column chromatography on DEAE-cellulose. Each activity results in formation of both glucosides, and the enzymes use UDP-glucose as the source of the glucose residue. A sensitive radiotracer assay was necessary to detect maximum specific activities of a few nmol h −1 g −1 of tissue. The results presented indicate that these enzymes are responsible for the formation of glucosides from BAP when it is fed to radish seedlings in aqueous solution, and may also form zeatin-7-glucoside, a naturally-occurring cytokinin.

Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa

The monooxygenase, p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) has been isolated and purified from Pseudomonas aeruginosa. The reaction catalysed is linked to the pathways for degradation of aromatic compounds by microorganisms. The enzyme has been quantitatively characterized in this paper for use in the mechanistic analysis of the protein by site-directed mutagenesis. This can be achieved when the results presented are used in combination with the information on the sequence and structure of the gene for this protein and the high-resolution crystallographic data for the protein from P. fluorescens. The protein is a dimer of identical sub-units in solution, and has one FAD per polypeptide with a monomeric molecular weight of 45,000. A full steady-state kinetic analysis was carried out at the optimum pH (8.0). A Vmax of 3750 min-1 at 25 degrees C was calculated, and the enzyme has a concerted-substitution mechanism, involving the substrates, NADPH, oxygen, and p-hydroxybenzoate. Extensive analyses of the reactions of reduced enzyme with oxygen were carried out. The quality of the data obtained confirmed the mechanisms of these reactions as proposed earlier by the authors for the enzyme from P. fluorescens. It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis. Thus, transient state kinetic analysis is an excellent tool to examine the role of amino acid residues in catalysis.