Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

Cloning, expression and characterization of a phosphoglucomutase/phosphomannomutase from sphingan-producing Sphingomonas sanxanigenens

Several strains of the genus Sphingomonas produce sphingans, extracellular polysaccharides used as thickeners, emulsifiers and gelling agents. The pgmG gene from Sphingomonas sanxanigenens, which encodes a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, was cloned and sequenced. The predicted amino acid sequence of the PgmG protein possessed 460 amino acids and a calculated molecular mass of 49.8 kDa, and it was 80 % identical to PGM/PMM from S. elodea. We overexpressed pgmG in Escherichia coli, and the purified protein displayed a K _m of 0.2 mM and a V _max of 1.3 μmol min^?1 mg^?1 with glucose 1-phosphate as substrate. The catalytic efficiency (K _cat/K _m) of PgmG was about 15-fold higher for glucose 1-phosphate than for mannose 1-phosphate. Overexpression of pgmG in S. sanxanigenens resulted in a 17 ± 0.3 % increase in sphingan production to ~12.5 g l^?1.     

The inhibition of phosphoglucomutase by beryllium

1. The inhibition of phosphoglucomutase by beryllium has been examined. 2. Inhibition by beryllium does not occur unless a complex-forming agent such as cysteine or imidazole is present. It is therefore similar to activation by magnesium. 3. The inhibition is progressive and the rate follows first-order kinetics, which may be defined by a bimolecular rate constant. 4. In the presence of magnesium the rate of inhibition is less. By using a fixed time for inhibition competition between beryllium and magnesium may be demonstrated. After inhibition has taken place, the addition of magnesium does not reverse it.     

Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.     

Genetic analysis of phosphomannomutase/phosphoglucomutase from<Emphasis Type="Italic"> Vibrio furnissii</Emphasis> and characterization of its role in virulence

The pmm gene from Vibrio furnissii, which encodes phosphomannomutase (PMM), was cloned and sequenced. The open reading frame consisted of 1,434 bp, encoding a polypeptide of 477 amino acids with a molecular mass of 53,325 Da. The predicted amino acid sequence of V. furnissii PMM showed high similarity with PMMs from other enteric bacteria, such as V. cholerae, Salmonella sp. and Escherichia coli. The PMM protein was overexpressed in E. coli as a His₆-tagged recombinant protein. The estimated apparent K_m and k_cat values of the purified recombinant protein for mannose 1-phosphate were about 60 M and 800 min^–1, respectively. To investigate the biochemical functions and the role of pmm in the virulence of V. furnissii, a pmm knock-out mutant was constructed by homologous recombination mutation. Under the various physical conditions, cell numbers of the wild-type and the mutant did not differ. Oral introduction of bacterial suspensions to a mouse model showed that the pmm-deficient mutant decreased in viability at the intestine. Microscopy of the isolated intestines from mice revealed significant damage after 3 days in intestinal mucosa infected with the wild-type as compared with the mutant. The pmm-deficient mutant caused a reduction of virulence in mice and the loss of O-antigen polysaccharide, and showed low resistance relative to the wild-type when incubated with normal human serum.     

Experimental and kinetic studies on methylene blue adsorption by coir pith carbon

Varying the parameters such as agitation time, dye concentration, adsorbent dose, pH and temperature carried out the potential feasibility of thermally activated coir pith carbon prepared from coconut husk for removal of methylene blue. Greater percentage of dye was removed with decrease in the initial concentration of dye and increase in amount of adsorbent used. Kinetic study showed that the adsorption of dye on coir pith carbon was a gradual process. Lagergren first-order, second-order, intra particle diffusion model and Bangham were used to fit the experimental data. Equilibrium isotherms were analysed by Langmuir, Freundlich, Dubnin-Radushkevich, and Tempkin isotherm. The adsorption capacity was found to be 5.87 mg/g by Langmuir isotherm for the particle size 250-500 microm. The equilibrium time was found to be 30 and 60 min for 10 and 20 mg/L and 100 min for 30, 40 mg/L dye concentrations, respectively. A maximum removal of 97% was obtained at natural pH 6.9 for an adsorbent dose of 100 mg/50 mL and 100% removal was obtained for an adsorbent dose of 600 mg/50 mL of 10 mg/L dye concentration. The pH effect and desorption studies suggest that chemisorption might be the major mode of the adsorption process. The change in entropy (DeltaS0) and heat of adsorption (DeltaH0) of coir pith carbon was estimated as 117.20 J/mol/K and 30.88 kJ/mol, respectively. The high negative value of change in Gibbs free energy indicates the feasible and spontaneous adsorption of methylene blue on coir pith carbon.     

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.     

Cell suspensions for kinetic studies of inhibitor action

Because of uniformity and small distances for transport, cell suspensions offer a system for rapid measurements of initial reactions of phytotoxic compounds. We had previously shown that a growth regulator, dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-L-gulonate) inhibits amino acid incorporation into proteins. Using Solanum nigrum suspension cultures, it was found that dikegulac rapidly inhibits amino acid uptake into cells, before inhibiting incorporation, with time points starting at a few minutes, and kinetics that can be extrapolated back to time zero. With more rapid kinetics this compound induces leakage of a preloaded dye. The rate of leakage was less with stationary cells in suspension, reiterating that they are more resistant to the effects of this compound. It was thus concluded that at the concentrations used, the first effect of dikegulac (or one very close to the first effect) is on the cell membrane.Abbreviation FDA fluorescein diacetate     

Mechanistic and kinetic studies of inhibition of enzymes

A graphical method for analyzing enzyme data to obtain kinetic parameters, and to identify the types of inhibition and the enzyme mechanisms, is described. The method consists of plotting experimental data as nu/(V0 - nu) vs 1/(I) at different substrate concentrations. I is the inhibitor concentration; V0 and nu are the rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate, and in the absence and presence of inhibitor, respectively. Complete inhibition gives straight lines that go through the origin; partial inhibition gives straight lines that converge on the 1-I axis, at a point away from the origin. For competitive inhibition, the slopes of the lines increase with increasing-substrate concentration; with noncompetitive inhibition, the slopes are independent of substrate concentration; with uncompetitive inhibition, the slopes of the lines decrease with increasing substrate concentrations. The kinetic parameters, Km, Ki, Ki', and beta (degree of partiality) can best be determined from respective secondary plots of slope and intercept vs substrate concentration, for competitive and noncompetitive inhibition mechanism or slope and intercept vs reciprocal substrate concentration for uncompetitive inhibition mechanism. Functional consequencs of these analyses are represented in terms of specific enzyme-inhibitor systems.     

Development of bioreactor systems with functional bio-carrier modified by disperse turquoise blue S-GL for disperse scarlet S-BWFL decolorization

The effect of redox mediator has been studied in details in the bio-decolorization processes, but there are little literatures about bioreactor systems with functional bio-carrier modified by redox mediator. Two different bioreactor configurations (bioreactor R1 with functional bio-carrier modified by disperse turquoise blue S-GL (as redox mediator) and bioreactor R2 with non-modified bio-carrier) were designed and tested for disperse scarlet S-BWFL decolorization by Halomonas sp. GYW (EF188281) in this study. Influencing factors such as co-substrate, temperature and pH were optimized through batch experiments. Compared to bioreactor R2, bioreactor R1 exhibited good decolorization efficiency and performance ability for the disperse scarlet S-BWFL decolorization, which showed higher decolorization efficiency (over 96% color removal with 0.8 g L(-1) dye concentration) and less hydraulic retention time to attain the same decolorization efficiency. The combinational technology of redox mediator and bio-carrier was a new bio-treatment concept and a great improvement for the application of redox mediator.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Structural analysis of the inhibition of thermolysin by an active-site-directed irreversible inhibitor

The mode of binding of the irreversible thermolysin inhibitor ClCH2CO-DL-(N-OH)Leu-OCH3 [Rasnick, D., & Powers, J.C. (1978) Biochemistry 17, 4363-4369] has been determined by X-ray crystallography at a resolution of 2.3 A and the structure of the covalent complex refined to give a crystallographic residual of 17.0%. This is the first such structural study of an active-site-directed covalent complex of a zinc protease. As anticipated by Rasnick and Powers, the inhibitor alkylates Glu-143 in the thermolysin active site, and the hydroxamic acid moiety coordinates the zinc ion. The formation of the covalent complex is associated with a significant shift in a segment of the polypeptide backbone in the vicinity of the active site. This conformational adjustment appears to be necessary to relieve steric hindrance which would otherwise prevent alkylation of Glu-143. It is suggested that this steric hindrance, which occurs for thermolysin but would not be expected for carboxypeptidase A, accounts for the previously inexplicable difference in reactivity of these two metalloproteases toward N-haloacetyl amino acids. The relevance of this steric hindrance to the mechanism of catalysis is discussed. In agreement with previous results [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506-2516], it appears that steric hindrance prevents the direct attack of Glu-143 on the carbonyl carbon of an extended substrate, therefore ruling out the anhydride pathway in thermolysin-catalyzed hydrolysis of polypeptide substrates and their ester analogues.     

Slow-tight binding inhibition of xylanase by an aspartic protease inhibitor: kinetic parameters and conformational changes that determine the affinity and selectivity of the bifunctional nature of the inhibitor

The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 +/- 1 x 10(-6) s(-1) and 5 +/- 0.5 x 10(-8) s(-1), respectively. The K(i) value for the formation of EI complex was 2.5 +/- 0.5 microm, whereas the overall inhibition constant K(i)* was 7 +/- 1 nm. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.