Kinetics of Sulfate and Acetate Uptake by Desulfobacter postgatei

The kinetics of sulfate and acetate uptake was studied in the sulfate-reducing bacterium Desulfobacter postgatei (DSM 2034). Kinetic parameters (K_m and V_max) were estimated from substrate consumption curves by resting cell suspensions with [³⁵S]sulfate and [¹⁴C]acetate. Both sulfate and acetate consumption followed Michaelis-Menten saturation kinetics. The half-saturation constant (K_m) for acetate uptake was 70 μM with cells from either long-term sulfate- or long-term acetate-limited chemostat cultures. The average K_m value for sulfate uptake by D. postgatei was about 200 μM. K_m values for sulfate uptake did not differ significantly when determined with cells derived either from batch cultures or sulfate- or acetate-limited chemostat cultures. Acetate consumption was observed at acetate concentrations of ≤1 μM, whereas sulfate uptake usually ceased at 5 to 20 μM. The results show that D. postgatei is not freely permeable to sulfate ions and further indicate that sulfate uptake is an energy-requiring process.     

Metabolic Activity of Fatty Acid-Oxidizing Bacteria and the Contribution of Acetate, Propionate, Butyrate, and CO2 to Methanogenesis in Cattle Waste at 40 and 60°C

The quantitative contribution of fatty acids and CO₂ to methanogenesis was studied by using stirred, 3-liter bench-top digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60°C under identical loading conditions (6 g of volatile solids per liter of reactor volume per day, 10-day retention time). In the thermophilic digestor, acetate turnover increased from a prefeeding level of 16 μM/min to a peak (49 μM/min) 1 h after feeding and then gradually decreased. Acetate turnover in the mesophilic digestor increased from 15 to 40 μM/min. Propionate turnover ranged from 2 to 5.2 and 1.5 to 4.5 μM/min in the thermophilic and mesophilic digestors, respectively. Butyrate turnover (0.7 to 1.2 μM/min) was similar in both digestors. The proportion of CH₄ produced via the methyl group of acetate varied with time after feeding and ranged from 72 to 75% in the mesophilic digestor and 75 to 86% in the thermophilic digestor. The contribution from CO₂ reduction was 24 to 29% and 19 to 27%, respectively. Propionate and butyrate turnover accounted for 20% of the total CH₄ produced. Acetate synthesis from CO₂ was greatest shortly after feeding and was higher in the thermophilic digestor (0.5 to 2.4 μM/min) than the mesophilic digestor (0.3 to 0.5 μM/min). Counts of fatty acid-degrading bacteria were related to their turnover activity.     

Nitrate Reduction in a Sulfate-Reducing Bacterium, Desulfovibrio desulfuricans, Isolated from Rice Paddy Soil: Sulfide Inhibition, Kinetics, and Regulation

From the second-highest dilution in a most-probable-number dilution series with lactate and sulfate as substrates and rice paddy soil as the inoculum, a strain of Desulfovibrio desulfuricans was isolated. In addition to reducing sulfate, sulfite, and thiosulfate, the strain also reduced nitrate to ammonia. The latter process was studied in detail, since the ability to reduce nitrate was strongly influenced by the presence of sulfide. Sulfide inhibited both growth on nitrate and nitrate reduction. A 70% inhibition of the nitrate reduction rate was obtained at 127 μM sulfide, and growth was inhibited by 50% at approximately 320 μM sulfide and was not detectable above 700 μM sulfide. In contrast, sulfate reduction was not affected at concentrations of up to 5 mM. After growth with sulfate, an induction period of 2 to 4 days was needed before nitrate reduction started. When nitrate and sulfate were present simultaneously, only sulfate was reduced, except when sulfate was present at very low concentrations (4 μM). At higher sulfate concentrations (500 μM), nitrate reduction was temporarily halted. The affinity for nitrate uptake was extremely high (K_m = 0.05 μM) compared with that for sulfate uptake (K_m = 5 μM). Thus, at low nitrate concentrations this bacterium is favored relative to denitrifiers (K_m = 1.8 to 13.7 μM) or other nitrate ammonifiers (e.g., Clostridium spp. [K_m = 500 μM]).     

Inhibition of the Fermentation of Propionate to Methane by Hydrogen, Acetate, and Propionate

Inhibition of the fermentation of propionate to methane and carbon dioxide by hydrogen, acetate, and propionate was analyzed with a mesophilic propionate-acclimatized sludge that consisted of numerous flocs (size, 150 to 300 μm). The acclimatized sludge could convert propionate to methane and carbon dioxide stoichiometrically without accumulating hydrogen and acetate in a propionate-minimal medium. Inhibition of propionate utilization by propionate could be analyzed by a second-order substrate inhibition model (shown below) given that the substrate saturation constant, K_s, was 15.9 μM; the substrate inhibition constant, K_i, was 0.79 mM; and the maximum specific rate of propionate utilization, q_m, was 2.15 mmol/g of mixed-liquor volatile suspended solids (MLVSS) per day: q_s = q_mS/[K_s + S + (S²/K_i)], where q_s is the specific rate of propionate utilization and S is the initial concentration of undissociated propionic acid. For inhibition by hydrogen and acetate to propionate utilization, a noncompetitive product inhibition model was used: q_s = q_m/[1 + (P/K_p)ⁿ], where P is the initial concentration of hydrogen or undissociated acetic acid and K_p is the inhibition constant. Kinetic analysis gave, for hydrogen inhibition, K_p(H2) = 0.11 atm (= 11.1 kPa, 71.5 μM), q_m = 2.40 mmol/g of MLVSS per day, and n = 1.51 and, for acetate inhibition, K_p(HAc) = 48.6 μM, q_m = 1.85 mmol/g of MLVSS per day, and n = 0.96. It could be concluded that the increase in undissociated propionic acid concentration was a key factor in inhibition of propionate utilization and that hydrogen and acetate cooperatively inhibited propionate degradation, suggesting that hydrogenotrophic and acetoclastic methanogens might play an important role in enhancing propionate degradation to methane and carbon dioxide.     

Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (K_m, 48 μM) to ectoine (K_m, 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (K_m, 63 μM) to betaine (K_m, 333 μM) and proline (K_m, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.     

Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (~20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K_m for NADP⁺ is ~37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.     

Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His₆-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX₂₄TX₁₃₁HX₁₀R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent K_m values were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparent K_m values for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAα-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.     

Kinetic Parameters of the Conversion of Methane Precursors to Methane in a Hypereutrophic Lake Sediment

The kinetic parameters K_m, V_max, T_t (turnover time), and v (natural velocity) were determined for H₂ and acetate conversion to methane by Wintergreen Lake sediment, using short-term (a few hours) methods and incubation temperatures of 10 to 14°C. Estimates of the Michaelis-Menten constant, K_m, for both the consumption of hydrogen and the conversion of hydrogen to methane by sediment microflora averaged about 0.024 μmol g⁻¹ of dry sediment. The maximal velocity, V_max, averaged 4.8 μmol of H₂ g⁻¹ h⁻¹ for hydrogen consumption and 0.64 μmol of CH₄ g⁻¹ h⁻¹ for the conversion of hydrogen to methane during the winter. Estimated natural rates of hydrogen consumption and hydrogen conversion to methane could be calculated from the Michaelis-Menten equation and estimates of K_m, V_max, and the in situ dissolved-hydrogen concentration. These results indicate that methane may not be the only fate of hydrogen in the sediment. Among several potential hydrogen donors tested, only formate stimulated the rate of sediment methanogenesis. Formate conversion to methane was so rapid that an accurate estimate of kinetic parameters was not possible. Kinetic experiments using [2-¹⁴C]acetate and sediments collected in the summer indicated that acetate was being converted to methane at or near the maximal rate. A minimum natural rate of acetate conversion to methane was estimated to be about 110 nmol of CH₄ g⁻¹ h⁻¹, which was 66% of the V_max (163 nmol of CH₄ g⁻¹ h⁻¹). A 15-min preincubation of sediment with 5.0 × 10⁻³ atm of hydrogen had a pronounced effect on the kinetic parameters for the conversion of acetate to methane. The acetate pool size, expressed as the term K_m + S_n (S_n is in situ substrate concentration), decreased by 37% and T_t decreased by 43%. The V_max remained relatively constant. A preincubation with hydrogen also caused a 37% decrease in the amount of labeled carbon dioxide produced from the metabolism of [U-¹⁴C]valine by sediment heterotrophs.     

Autotrophic, Hydrogen-Oxidizing, Denitrifying Bacteria in Groundwater, Potential Agents for Bioremediation of Nitrate Contamination

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent K_m of 0.45 to 0.60 μM and a V_max of 18.7 nmol cm^-3 h^-1 at 30°C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to Paracoccus denitrificans, a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the K_ms ranged from 0.30 to 3.32 μM, with V_maxs of 1.85 to 13.29 fmol cell^-1 h^-1. Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.     

C3larvin Toxin,an ADP-ribosyltransferase from Paenibacillus larvae

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD⁺ (K_m = 34 ± 12 μm) and RhoA (K_m = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (K_i = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins.     

Measurement of Nitrous Oxide Reductase Activity in Aquatic Sediments

Denitrification in aquatic sediments was measured by an N₂O reductase assay. Sediments consumed small added quantities of N₂O over short periods (a few hours). In experiments with sediment slurries, N₂O reductase activity was inhibited by O₂, C₂H₂, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 μM) did not influence activity, and moderate levels (about 150 μM) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N₂O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater (K_m = 2.17 μM), estuarine (K_m = 14.5 μM), and alkaline-saline (K_m = 501 μM) environments. An in situ assay was devised in which a solution of N₂O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N₂O per m² per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N₂O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N₂O per m² per h made with the acetylene block assay.     

Two Nucleotide Transport Proteins in Chlamydia trachomatis, One for Net Nucleoside Triphosphate Uptake and the Other for Transport of Energy

The genome of Chlamydia trachomatis, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1_Ct and Npt2_Ct (nucleoside phosphate transporters 1 and 2 of C. trachomatis), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium Rickettsia prowazekii at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in Escherichia coli to study their biochemical properties. His₁₀-Npt1_Ct catalyzed ATP and ADP transport in an exchange mode. The apparent K_m values were 48 (ATP) and 39 (ADP) μM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His₁₀-Npt2_Ct transported all four ribonucleoside triphosphates with apparent K_m values of 31 μM (GTP), 302 μM (UTP), 528 μM (CTP), and 1,158 μM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m-chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2_Ct. This observation indicated that His₁₀-Npt2_Ct acts as a nucleosidetriphosphate/H⁺ symporter energized by the proton motive force across the Escherichia coli cytoplasmic membrane. We conclude that Npt1_Ct provides chlamydiae with energy whereas Npt2_Ct catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.     

Product Inhibition of Butyrate Metabolism by Acetate and Hydrogen in a Thermophilic Coculture

Studies on product inhibition of a thermophilic butyrate-degrading bacterium in syntrophic association with Methanobacterium thermoautotrophicum showed that a gas phase containing more than 2 × 10⁻² atm (2.03 kPa) of hydrogen prevented growth and butyrate consumption, while a lower hydrogen partial pressure of 1 × 10⁻³ to 2 × 10⁻² atm (0.1 to 2.03 kPa) gradually inhibited the butyrate consumption of the coculture. No inhibition of butyrate consumption was found on the addition of 0.75 × 10⁻³ atm (76 Pa) of hydrogen to the gas phase. A slight inhibition of butyrate consumption by the coculture occurred at an acetate concentration of 16.4 mM. Inhibition gradually increased with increasing acetate concentration up to 81.4 mM, when complete inhibition of butyrate consumption occurred. When the culture contained an acetate-utilizing methanogen in addition to M. thermoautotrophicum, the inhibition of the triculture by acetate was gradually reversed as the acetate concentration was lowered by the aceticlastic methanogen. The results show that optimal growth conditions for the thermophilic butyrate-degrading bacterium depend on both hydrogen and acetate removal.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

In Intact Cone Photoreceptors,a Ca2+-dependent,Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods

We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K _1/2, also increases as Ca²⁺ concentration decreases. In electropermeabilized cones, K _1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca²⁺, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K _1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca²⁺ and 15.3 ± 4.5 μM in its absence. The Ca²⁺-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K _1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca²⁺ and 7.2 ± 1.2 μM in its absence. The Ca²⁺-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca²⁺-dependent change in sensitivity in cones is half-maximal at K _Ca = 286 ± 66 nM Ca²⁺. In rods, by contrast, K _Ca is ∼50 nM Ca²⁺. The difference in magnitude and Ca²⁺ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.     

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

1. Cholesteryl 3β-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K_m for cholesteryl sulphate is 500μm and for free cholesterol 50μm under the same conditions. 3. Cholesteryl 3β-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K_i for cholesteryl phosphate 28μm, for cholesteryl sulphate 110μm, for cholesteryl acetate 65μm) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20α-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K_i for pregnenolone is 130μm and K_i for 20α-hydroxycholesterol is 17μm. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K_i16μm). A number of other Δ⁵-3β-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3β-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20α-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3β-Hydroxychol-5-enoic acid, 3α-hydroxy-5β-cholanic acid and 3β-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3β-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K_m and V_max values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 μM. The K_m and V_max values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 μM. The apparent K_m for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K_m for H₂ uptake by cultures of Methanobacterium formicicum was 6 μM dissolved H₂. Formate and H₂ were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H₂ uptake was severely depressed when formate was above saturation and the dissolved H₂ was below 6 μM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.     

The activities and kinetic properties of purine phosphoribosyltransferases in developing mouse liver

1. The total activity of adenine phosphoribosyltransferase/liver of mice remained constant from 1 to 16 days after birth despite a fourfold increase in liver weight. The total activity of this enzyme increased fivefold from 16 to 36 days and then remained relatively constant at least until 96 days after birth. Total hypoxanthine-phosphoribosyltransferase activity/liver steadily increased between 1 and 57 days after birth. 2. The mean K_m of 5-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase was 10·1μm between 3 and 11 days, at 64 days and at 96 days after birth. Between 17 and 51 days the mean K_m value was 3·0μm. The K_m of 5-phosphoribosyl pyrophosphate with hypoxanthine phosphoribosyltransferase remained constant at 28·2μm between 2 and 64 days. 3. Adenine-phosphoribosyltransferase activity was stimulated between 15 and 83% by 60μm-ATP when extracts were made between 3 and 11 days, at 64 days or at 96 days after birth. Between 17 and 51 days ATP had little stimulatory effect on the activity of this enzyme. 4. AMP competed with 5-phosphoribosyl pyrophosphate in the reaction catalysed by adenine phosphoribosyltransferase. Liver extracts containing enzyme with a low value of K_m for 5-phosphoribosyl pyrophosphate (3μm) had a K_m/K_i ratio approximately half that of extracts with a high value of K_m (10μm). 5. The results indicate that two different forms of adenine phosphoribosyltransferase can exist in mouse liver at different stages of development. The physiological significance of these findings is discussed.     

Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.     

Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala^*Gly-Pro-Ile-Gly (k_cat, 5.41 s⁻¹; K_m, 32 μM) and Met-Gly-Pro-Arg^*Gly-Phe-Pro-Gly-Ser (k_cat, 351 s⁻¹; K_m, 214 μM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.