Influence of environmental factors on reductive bioprecipitation of uranium by sulfate reducing bacteria

Microbial reduction of soluble uranyl [U (VI)] to insoluble uraninite by sulfate reducing bacteria (SRB) is a promising remediation strategy for uranium-contaminated groundwater. Effects of environmental factors, including pH and coexisting ions, on U (VI) bioreduction processes (UBP) remain unknown. Anaerobic batch experiments were performed to evaluate impact on UBP. Kinetic investigations with varied pH demonstrated that U (VI) was reduced mostly within 48 h. The bioprecipitation yields depended strongly on pH, increasing from 12.9% to 99.4% at pH 2.0 and 6.0, respectively. Sulfate concentration 4000 mg l⁻¹ did not affect UBP; however, sulfate concentration 5000 mg l⁻¹ significantly slowed UBP. Biogenic H₂S produced during sulfate reduction was not directly involved in UBP. At 20 mg l⁻¹ Zn or 10 mg l⁻¹ Cu, no UBP inhibition was observed and uraninite was detected in metal sulfide precipitate. However, 25 mg l⁻¹ Zn or 15 mg l⁻¹ Cu stopped UBP completely. Cu toxicity mechanism probably differed from Zn. The ability to reduce U (VI) was lost permanently with exposure to 15 mg l⁻¹ Cu, but not for Zn 25 mg l⁻¹. No uraninite could be detected before nitrate removal, suggesting nitrate strongly inhibited UBP, which may possibly be related to denitrification intermediates controlling the solution redox potential.     

The molybdate binding protein Mop from Haemophilus influenzae--biochemical and thermodynamic characterisation

The protein Mop from Haemophilus influenzae is a member of the molbindin family of proteins. Using isothermal titration calorimetry (ITC), Mop was observed to bind molybdate at two distinct sites with a stoichiometry of 8 mol molybdate per Mop hexamer. Six moles of molybdate bound endothermically at high affinity sites (K(a)=8.5 x 10(7)M(-1)), while 2 mol of molybdate bound exothermically at lower affinity sites (K(a)=3.7 x 10(7)M(-1)). Sulphate was also found to bind weakly at the higher affinity sites. ITC revealed that the affinity of molybdate binding to the endothermic site decreased with increasing pH and was accompanied by the transfer from the buffer to the protein of one proton per Mop monomer. These kinetic and thermodynamic results are interpreted with reference to molbindin crystal structures and data concerning molbindin binding affinities. Mop binds molybdate with high specificity, capacity, and affinity which indicates that Mop has a role as an intracellular molybdate binding protein involved in oxyanion homeostasis.     

The role of specific β–γ subunit interactions in oxyanion stimulation of the MgATP hydrolysis of a hybrid photosynthetic F1-ATPase

Pairs of cysteine residues were introduced into the twisted N- and C-terminal helices of the γ subunit of the chloroplast F₁-ATPase to test, via disulfide cross-linking, potential inter-helical movements involved in catalysis of ATP hydrolysis. The extent of disulfide cross-linking was determined by estimating the amount of free sulfhydryl available for labeling with fluoresceinyl maleimide before and after cross-linking. Significant disulfide formation (50–75%) was observed between cysteines introduced at positions 30 and 31 in the N-terminal helix and 276 and 278 in the C-terminal helix. Cross-linking had no apparent effect on catalysis, therefore eliminating the involvement of large-scale inter-helical movements within this region of the γ subunit in cooperative ATP hydrolysis. However, the presence of the two cysteines together in the γV31C/A276C double mutant, irrespective of whether or not they were cross-linked together, lowered the MgATPase activity by more than 70% and completely eliminated the well-known activating effect of the oxyanion sulfite. The CaATPase activity was unaffected. Similar but less pronounced effects were seen with the γK30C/A276C double mutant. The results indicate that residues at or near positions 31 and 276 within the twisted helical pair of the γ subunit are required to overcome Mg²⁺ inhibition of ATP hydrolysis. These residues are likely to be involved in forming a point of contact between the γ and β subunits that is responsible for this effect.     

Aldol additions with mutant lipase: analysis by experiments and theoretical calculations

A Ser105Ala mutant of Candida antarctica lipase B has previously been shown to catalyze aldol additions. Quantum chemical calculations predicted a reaction rate similar to that of natural enzymes, whereas experiments showed a much lower reaction rate. Molecular dynamics simulations, presented here, show that the low reaction rate is a consequence of the low frequencies of near attack complexes in the enzyme. Equilibrium was also considered as a reason for the slow product formation, but could be excluded by performing a sequential reaction to push the reaction towards product formation. In this paper, further experimental results are also presented, highlighting the importance of the entire active site for catalysis.     

Ionisations within a subtilisin–glyoxal inhibitor complex

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K_i=2.3±0.2 μM at pH 7.0 and 25 °C. Using Z-Ala-Pro-[2-¹³C]Phe-glyoxal we have detected a signal at 107.3 ppm by ¹³C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the ¹³C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK_a<3. This is the first observation of oxyanion formation in a reversible subtilisin–inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK_a of ～6.5 in the free enzyme and on a pK_a<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol^?1. We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.     

Hepatitis C NS3 protease inhibition by peptidyl-alpha-ketoamide inhibitors: kinetic mechanism and structure

A series of novel peptidyl-alpha-ketoamide compounds were evaluated as inhibitors of the deltaNS3-NS4A serine protease from the hepatitis C virus. These peptidyl-alpha-ketoamide inhibitors with Ki values ranging from 0.17 nM to 5.6 microM exhibited slow-binding inhibition. Kinetic studies established one-step kinetic mechanisms and dissociation rate constants in the 3-7 x 10(-5) s(-1) range for these compounds. The association rate constants, which ranged from 10 to 263,000 M(-1) s(-1), were responsible for the greater than four order of magnitude overall binding affinity range exhibited by this series. An X-ray crystal structure of a protease-inhibitor complex revealed an unusual interaction between the oxyanion of the adduct and the protein as well as a significant movement in the S1' region of the protein loop comprising residues 35-42. These results are quite different from peptidyl-alpha-ketoacid inhibition of HCV protease, which reportedly undergoes no notable conformational changes and proceeds with a two-step slow-binding kinetic mechanism.     

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma)

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.     

Development of a viologen-based microtiter plate assay for the analysis of oxyanion reductase activity: application to the membrane-bound selenate reductase from Enterobacter cloacae SLD1a-1

The membrane-bound selenate reductase of Enterobacter cloacae SLD1a-1 is purified in low yield and has relatively low activity in the pure form compared to that of other oxyanion reductases, such as the membrane-bound and periplasmic nitrate reductases. A microtiter plate assay based on the original quartz cuvette viologen assay of Jones and Garland (R.W. Jones, P.B. Garland, Biochem. J 164 (1977) 199-211) was developed specifically for analysis of such low-abundant, labile oxyanion reductases. The plate assay detects the enzyme-dependent reoxidation of reduced methyl viologen spectrophotometrically at 600 nm. The assay is quick, uses a minimal sample volume (<5 microl), can simultaneously test a range of alternative substrates, and permits activity measurements on multiple samples. We demonstrate the accuracy and versatility of the microtiter plate assay by application to the kinetic analysis, inhibition, and pH optimization of the membrane-bound selenate reductase from E. cloacae SLD1a-1. Results show that the membrane-bound selenate reductase has optimum activity at pH approximately 8 and its active site is able to accommodate larger inhibitory complexes resulting in mixed-type inhibition, in the presence of selenate and potassium thiocyanate.     

Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion

Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme⿿s enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50 °C, after 12⿿16 h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.