Structural and functional analyses reveal that Staphylococcus aureus antibiotic resistance factor HmrA is a zinc-dependent endopeptidase.

HmrA is an antibiotic resistance factor of methicillin-resistant Staphylococcus aureus. Molecular analysis of this protein revealed that it is not a muramidase or ß-lactamase but a nonspecific double-zinc endopeptidase consisting of a catalytic domain and an inserted oligomerization domain, which probably undergo a relative interdomain hinge rotation upon substrate binding. The active-site cleft is located at the domain interface. Four HmrA protomers assemble to a large ∼170-kDa homotetrameric complex of 125 Å. All four active sites are fully accessible and ∼50-70 Å apart, far enough apart to act on a large meshwork substrate independently but simultaneously. In vivo studies with four S. aureus strains of variable resistance levels revealed that the extracellular addition of HmrA protects against loss of viability in the presence of oxacillin and that this protection depends on proteolytic activity. All of these results indicate that HmrA is a peptidase that participates in resistance mechanisms in vivo in the presence of ß-lactams. Furthermore, our results have implications for most S. aureus strains of known genomic sequences and several other cocci and bacilli, which harbor close orthologs. This suggests that HmrA may be a new widespread antibiotic resistance factor in bacteria.

Proteolysis of peptide dendrimers.

The ability of proteins and peptides to undergo proteolysis is essential to their biological function. Herein, we report the first detailed study of the protease reactivity of peptide dendrimers. Dendrimers are regularly ramified, tree-like synthetic macromolecules with promising application in technology and medicine. Using trypsin and alpha-chymotrypsin cleavage sites as models, we show that the protease reactivity of peptide dendrimers can be controlled by the degree of branching. Dendrimers with two or three amino acids between branching points were readily cleaved by trypsin irrespective of the position of the reactive sequence within the dendrimers, for example in D1, (Ac-Gly-Phe-Pro)4(Dap-Hyp-Arg[downward arrow]Met)2Dap-Ser-Gly-betaAla-NH2, and D12, (Ac-Ser-Ala)8(Dap-Ala-Arg[downward arrow])4(Dap-Ala-Asp)2Dap-Phe-Ala-Lys*-NH2 (Dap: (S)-2,3-diaminopropionic acid branching point, Hyp: hydroxyproline, Lys*: FITC-labeled lysine, [downward arrow]: cleavage site). On the other hand cleavage was blocked in more compact dendrimers with only one amino acid between branching points, for example in D18B, (Ac-Glu)8(Dap-Phe)4(Dap-Arg)2Dap-Leu-NH2). The control of proteolysis by topology provides a novel possibility to tune the biological properties of peptide dendrimers not available in linear peptides, and should be generally useful for their use as functional biomolecule analogues, for example, in the context of drug delivery applications.

Enzyme assays.

Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

On-bead cyclization in a combinatorial library of 15,625 octapeptides.

Combinatorial peptide libraries prepared by split-and-mix synthesis on solid support can be decoded by amino acid analysis (AAA) using the TAGSFREE method, which assigns variable amino acids to 'unique pair' positions. The method was used here to investigate on-bead cyclization in a library of 15,625 octapeptides X(8)X(7)X(6)X(5)X(4)-Lys-X(2)-glu(beta-Ala-beta-Ala-TentaGel Macrobead)-OAllyl, anchored via the side-chain carboxylate of the d-glutamate. Cyclization was carried out by amide bond formation between the free N-terminus and the alpha-carboxyl group of d-glutamate after selective removal of the Fmoc and allyl protecting groups, and followed using the TNBS test for free amines. Fast-cyclizing sequences often contained a turn element, in particular Ala-(Asp/Thr)-Pro at X(8)-X(7)-X(6), and phenylalanine at X(2). Slow-cyclizing sequences contained predominantly basic and polar residues, in particular Arg-His-Ser at X(7)-X(6)-X(5) and threonine at X(8). Fast-cyclizing sequences gave higher preparative yields of cyclic peptides (22-26% purified yields) than slow-cyclizing sequences (6-8%), showing that fast reaction is associated with efficient cyclization. This experiment demonstrates the first use of a TAGSFREE library of cyclic peptides.

A cyclodecapeptide ligand to vitamin B12.

Libraries of cyclic decapeptides were screened with vitamin B(12) derivatives to give cyclic peptide ligands incorporating histidine and cysteine as coordinating residues and negatively charged amino acids. Two hits, cyclo-(HisAspGluProGlyIleAlaThrProdGln) and cyclo-(ValAspGluProGlyGluAspCysProdGln) were resynthesized in good yields for solution experiments. The peptides bind aquocobalamin with coordination of His or Cys to the cobalt with high affinities (K(a) approximately 10(5) M(-1)). Additional interactions between the peptide side chains and the vitamin B(12) corrin moiety were determined by studying the (1)H NMR solution structure. The cyclopeptide-cobalamin complex with the histidine residue showed enhanced stability towards cyanide exchange, demonstrating the shielding effect of the ligand on the metal center.

Bead diffusion assay for discovering antimicrobial cyclic peptides.

A combinatorial library of 15,536 cyclic decapeptide analogues of tyrocidine A and gramicidin S was prepared on photocleavable TentaGel beads. The beads were photolyzed without solvent, and spread onto an agar plate inoculated with bacterial lawn. Clear zones were formed around beads carrying antimicrobially active peptides such as E18 c(kVOrnLfThiYOrnLq), which inhibited growth of B. subtilis and selected MRSA strains.

Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test.

The adrenaline test for enzymes is a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products such as 1,2-diols and 1,2-aminoalcohols by back-titration of the oxidant with adrenaline to produce adrenochrome as an easily detectable red product. The test uses commercial reagents and is suitable for screening the activity of various hydrolases. It is demonstrated here for testing epoxide hydrolases, lipases and esterases, and for activity fingerprinting of these enzymes across substrate series. The complete assay requires 2-3 h.