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PubMed2003

Characterization of a novel Ser-cisSer-Lys catalytic triad in comparison with the classical Ser-His-Asp triad.

Amidase signature family enzymes, which are widespread in nature, contain a newly identified Ser-cisSer-Lys catalytic triad in which the peptide bond between Ser131 and the preceding residue Gly130 is in a cis configuration. In order to characterize the property of the novel triad, we have determined the structures of five mutant malonamidase E2 enzymes that contain a Cys-cisSer-Lys, Ser-cisAla-Lys, or Ser-cisSer-Ala triad or a substitution of Gly130 with alanine. Cysteine cannot replace the role of Ser155 due to a hyper-reactivity of the residue, which results in the modification of the cysteine to cysteinyl sulfinic acid, most likely inside the expression host cells. The lysine residue plays a structural as well as a catalytic role, since the substitution of the residue with alanine disrupts the active site structure completely. The two observations are in sharp contrast with the consequences of the corresponding substitutions in the classical Ser-His-Asp triad. Structural data on the mutant containing the Ser-cisAla-Lys triad convincingly suggest that Ser131 plays an analogous catalytic role as the histidine of the Ser-His-Asp triad. The unusual cis configuration of Ser131 appears essential for the precise contacts of this residue with the other triad residues, as indicated by the near invariance of the preceding glycine residue (Gly130), structural data on the G130A mutant, and by a modeling experiment. The data provide a deep understanding of the role of each residue of the new triad at the atomic level and demonstrate that the new triad is a catalytic device distinctively different from the classical triad or its variants.

Amidohydrolases↗

PubMed1995

Analysis of the role of the KMSKS loop in the catalytic mechanism of the tyrosyl-tRNA synthetase using multimutant cycles.

A mobile loop in tyrosyl-tRNA synthetase, which corresponds to the KMSKS signature sequence of class I aminoacyl-tRNA synthetases, destabilizes the E.Tyr.ATP complex but stabilizes the following E.[Tyr-ATP]not equal to transition state for the formation of E.Tyr-AMP. Three amino acid residues in the mobile loop, K230, K233, and T234, are known to be primarily responsible for these effects. We now analyze the network of interactions between these three amino acids using multiple mutant free energy cycles. The complete characterization of the coupling energies within the mobile loop allows each of the steps leading to the formation of the transition state complex to be dissected into its energetic components. In particular, it is found that, in the absence of a functional mobile loop, there is synergistic coupling between the tyrosine and ATP substrates (i.e., each enhances the binding affinity of the other) which stabilizes the E.Tyr.ATP intermediate preceding the transition state complex. Thus, the mobile loop disrupts the synergism between the ATP and tyrosine substrates, using the ATP binding energy to stabilize the transition state for the reaction. Whereas the net effect of the mobile loop in the E.Tyr.ATP complex results from several conflicting side chain interactions that tend to offset each other, conflicting interactions in the E.[Tyr-ATP]not equal to transition state complex have been minimized and stabilizing pairwise interactions between the K230, K233, and T234 side chains are optimized. The tight coupling between the side chains of K230, K233, and T234 suggests that the mobile loop adopts a highly constrained conformation during formation of the transition state complex. These results quantitatively demonstrate the importance of side chain interactions in enzyme catalysis and illustrate the use of binding energy to stabilize the transition state of a reaction and the presence of unfavorable interactions to destabilize the ground state.

Adenosine Triphosphate↗

PubMed2005

VirR, a response regulator critical for Listeria monocytogenes virulence.

Signature-tagged mutagenesis (STM) was used to identify new genes involved in the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the mutants isolated by this technique had the transposon inserted in virR, a gene encoding a putative response regulator of a two-component system. Deletion of virR severely decreased virulence in mice as well as invasion in cell-culture experiments. Using a transcriptomic approach, we identified 12 genes regulated by VirR, including the dlt-operon, previously reported to be important for L. monocytogenes virulence. However, a strain lacking dltA, was not as impaired in virulence as the DeltavirR strain, suggesting a role in virulence for other members of the vir regulon. Another VirR-regulated gene is homologous to mprF, which encodes a protein that modifies membrane phosphatidyl glycerol with l-lysine and that is involved in resistance to human defensins in Staphylococcus aureus. VirR thus appears to control virulence by a global regulation of surface components modifications. These modifications may affect interactions with host cells, including components of the innate immune system. Surprisingly, although controlling the same set of genes as VirR, the putative cognate histidine kinase of VirR, VirS, encoded by a gene located three genes downstream of virR, was shown not to be essential for virulence. By monitoring the activity of VirR with a GFP reporter construct, we showed that VirR can be activated independently of VirS, for example through a mechanism involving variations in the level of intracellular acetyl phosphate. In silico analysis of the VirR-regulated promoters revealed a VirR DNA-binding consensus site and specific interaction between purified VirR protein and this consensus sequence was demonstrated by gel mobility shift assays. This study identifies a second key virulence regulon in L. monocytogenes, after the prfA regulon.

Animals↗