Bioaccumulation of palladium by Desulfovibrio fructosivorans wild-type and hydrogenase-deficient strains.

Wild-type Desulfovibrio fructosivorans and three hydrogenase-negative mutants reduced Pd(II) to Pd(0). The location of Pd(0) nanoparticles on the cytoplasmic membrane of the mutant retaining only cytoplasmic membrane-bound hydrogenase was strong evidence for the role of hydrogenases in Pd(0) deposition. Hydrogenase activity was retained at acidic pH, shown previously to favor Pd(0) deposition.

Biocatalysts for fuel cells: efficient hydrogenase orientation for H2 oxidation at electrodes modified with carbon nanotubes.

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H(2) oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H(2) oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials.

Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments.

Enzymes MurD, MurE, MurF, folylpolyglutamate synthetase and cyanophycin synthetase, which belong to the Mur synthetase superfamily, possess an invariant lysine residue (K198 in the Escherichia coli MurD numbering). Crystallographic analysis of MurD and MurE has recently shown that this residue is present as a carbamate derivative, a modification presumably essential for Mg(2+) binding and acyl phosphate formation. In the present work, the importance of the carbamoylated residue was investigated in MurD, MurE and MurF by site-directed mutagenesis and chemical rescue experiments. Mutant proteins MurD K198A/F, MurE K224A and MurF K202A, which displayed low enzymatic activity, were rescued by incubation with short-chain carboxylic acids, but not amines. The best rescuing agent was acetate for MurD K198A, formate for K198F, and propionate for MurE K224A and MurF K202A. In the last of these, wild-type levels of activity were recovered. A complementarity between the volume of the residue replacing lysine and the length of the carbon chain of the acid was noted. These observations support a functional role for the carbamate in the three Mur synthetases. Experiments aimed at recovering an active enzyme by introducing an acidic residue in place of the invariant lysine residue were also undertaken. Mutant protein MurD K198E was weakly active and was rescued by formate, indicating the necessity of correct positioning of the acidic function with respect to the peptide backbone. Attempts at covalent rescue of mutant protein MurD K198C failed because of its lack of reactivity towards haloacids.

Role of the ortholog and paralog amino acid invariants in the active site of the UDP-MurNAc-L-alanine:D-glutamate ligase (MurD).

To evaluate their role in the active site of the UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) from Escherichia coli, 12 residues conserved either in the Mur superfamily [Eveland, S. S., Pompliano, D. L., and Anderson, M. S. (1997) Biochemistry 36, 6223-6229; Bouhss, A., Mengin-Lecreulx, D., Blanot, D., van Heijenoort, J., and Parquet, C. (1997) Biochemistry 36, 11556-11563] or in the sequences of 26 MurD orthologs were submitted to site-directed mutagenesis. All these residues lay within the cleft of the active site of MurD as defined by its 3D structure [Bertrand, J. A., Auger, D., Fanchon, E., Martin, L., Blanot, D., van Heijenoort, J., and Dideberg, O. (1997) EMBO J. 16, 3416-3425]. Fourteen mutant proteins (D35A, K115A, E157A/K, H183A, Y194F, K198A/F, N268A, N271A, H301A, R302A, D317A, and R425A) containing a C-terminal (His)(6) extension were prepared and their steady-state kinetic parameters determined. All had a reduced enzymatic activity, which in many cases was very low, but no mutation led to a total loss of activity. Examination of the specificity constants k(cat)/K(m) for the three MurD substrates indicated that most mutations affected both the binding of one substrate and the catalytic process. These kinetic results correlated with the assigned function of the residues based on the X-ray structures.

Formation of adenosine 5'-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli.

The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5'-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.