Involvement of a Novel Class C Beta-Lactamase in the Transglutaminase Mediated Cross-Linking Cascade of Streptomyces mobaraensis DSM 40847

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained K_m and k_cat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s^-1, respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (K_i of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn²⁺-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.     

Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis

The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA-mediated carbon catabolite regulation (CCR) in Bacillus subtilis. Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements (cre). We present a quantitative analysis of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance (SPR) revealing small and similar equilibrium constants of 4.8 +/- 0.4 microm for HPrSerP-CcpA and 19.1 +/- 2.5 microm for CrhP-CcpA complex dissociation. Forty millimolar fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc6-P) increases the affinity of HPrSerP to CcpA at least twofold, but have no effect on CrhP-CcpA binding. Saturation of binding of CcpA to cre as studied by fluorescence and SPR is dependent on 50 microm of HPrSerP or > 200 microm CrhP. The rate constants of HPrSerP-CcpA-cre complex formation are k(a) = 3 +/- 1 x 10(6) m(-1).s(-1) and k(d) = 2.0 +/- 0.4 x 10(-3).s(-1), resulting in a K(D) of 0.6 +/- 0.3 nm. FBP and Glc6-P stimulate CcpA-HPrSerP but not CcpA-CrhP binding to cre. Maximal HPrSerP-CcpA-cre complex formation in the presence of 10 mm FBP requires about 10-fold less HPrSerP. These data suggest a specific role for FBP and Glc6-P in enhancing only HPrSerP-mediated CCR.     

A novel transglutaminase substrate from Streptomyces mobaraensis inhibiting papain-like cysteine proteases

Transglutaminase from Streptomyces mobaraensis is an enzyme of unknown function that cross-links proteins to high molecular weight aggregates. Previously, we characterized two intrinsic transglutaminase substrates with inactivating activities against subtilisin and dispase. This report now describes a novel substrate that inhibits papain, bromelain, and trypsin. Papain was the most sensitive protease; thus, the protein was designated Streptomyces papain inhibitor (SPI). To avoid transglutaminase-mediated glutamine deamidation during culture, SPI was produced by Streptomyces mobaraensis at various growth temperatures. The best results were achieved by culturing for 30-50 h at 42 degrees C, which yielded high SPI concentrations and negligibly small amounts of mature transglutaminase. Transglutaminasespecific biotinylation displayed largely unmodified glutamine and lysine residues. In contrast, purified SPI from the 28 degrees C culture lost the potential to be cross-linked, but exhibited higher inhibitory activity as indicated by a significantly lower Ki (60 nM vs. 140 nM). Despite similarities in molecular mass (12 kDa) and high thermostability, SPI exhibits clear differences in comparison with all members of the wellknown family of Streptomyces subtilisin inhibitors. The neutral protein (pI of 7.3) shares sequence homology with a putative protein from Streptomyces lavendulae, whose conformation is most likely stabilized by two disulfide bridges. However, cysteine residues are not localized in the typical regions of subtilisin inhibitors. SPI and the formerly characterized dispase-inactivating substrate are unique proteins of distinct Streptomycetes such as Streptomyces mobaraensis. Along with the subtilisin inhibitory protein, they could play a crucial role in the defense of vulnerable protein layers that are solidified by transglutaminase.     

Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features.     

Enzymic preparation of protein G-peroxidase conjugates catalysed by transglutaminase

Transglutaminases (TGases, EC 2.3.2.13) have proved to be valuable enzymes for site-directed protein coupling via N(epsilon)-(gamma-L-glutamyl)lysine bonds. Their use in conjugate synthesis would overcome many problems caused by chemical reagents. In this approach, we show for the first time that two proteins with different functionalities, namely soybean peroxidase and protein G, can be cross-linked by bacterial TGase with retention of their activities. Soybean peroxidase and protein G were chosen for the enzymic preparation of a bifunctional conjugate among a series of other TGase substrates detected by enzymic incorporation of small fluorescent or biotinylated molecules. The highest yields of conjugate were obtained with a 15-fold excess of peroxidase in phosphate buffer, pH 7.0. Size exclusion chromatography enabled both purification of the conjugates and recovery of the starting materials. Analysis of bifunctionality revealed the coupling of protein G with an average of three peroxidase molecules.     

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI_p), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI_p exhibits a rigid, thermo‐resistant double‐psi‐beta‐barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln‐6 as the only amine acceptor site on SPI_p accessible for MTG. Substitution of Lys‐7 demonstrated that small and hydrophobic residues in close proximity to Gln‐6 favor MTG‐mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI_p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG‐substrates used in biomedical applications.     

Significance of Cuscutain,a cysteine protease from <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>, in host-parasite interactions

Background  

Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified.     

The transglutaminase activating metalloprotease inhibitor from Streptomyces mobaraensis is a glutamine and lysine donor substrate of the intrinsic transglutaminase

Transglutaminase (TGase) from Streptomyces mobaraensis is an extra-cellular enzyme that cross-links proteins to high molecular weight aggregates. Screening for intrinsic substrates now revealed the dual Streptomyces subtilisin inhibitor-like inhibitor Streptomyces subtilisin and transglutaminase activating metalloprotease (TAMEP) inhibitor (SSTI), equally directed against subtilisin and the TGase activating metalloprotease TAMEP, is both a glutamine and a lysine donor protein. Reactivity of glutamines is lost during culture, most likely by TGase mediated deamidation, and, accordingly, cross-linking only occurred if SSTI from early cultures was used. Interestingly, release of buried endo-glutamines by the lipoamino acid N-lauroylsarcosine could restore SSTI reactivity. Formation of lipoamino acids by Streptomycetes suggests such compounds could also modulate in vivo TGase mediated SSTI cross-linking.     

A sensitive fluorometric assay for tissue transglutaminase

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).