Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

A novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureus SspB) and culture supernatants (Porphyromonas gingivalis, Bacillus anthracis). Further, growth of Bacillus anthracis, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholerae was completely inhibited by 10 M SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria. P apain (EC 3.4.22.2) belongs to clan A of the cysteine protease enzymes and is the eponym for the C1 family of proteases, which comprises a large number of endopeptidases, although fewer exopeptidases. All members of the papain family contain cysteine and histidine at their active site, forming a catalytic dyad. Papain and related cysteine proteases are widely distributed in the plant kingdom and are believed to act as virulence/defense factors for both hosts and pathogens (1). Cysteine proteases are also found in bacteria and are known to be virulence factors involved in bacterial pathogenicity (2). Further, papain-like hydrolases are involved in peptidoglycan turnover in both Gram-negative and Gram-positive bacteria (3, 4), with disruption of amide-hydrolyzing autolysins in Bacillus subtilis, leading to defective cell wall division and thereby affecting bacterial viability (5). More recently, the growing resistance of microorganisms to conventionally used antibiotics has meant that cysteine proteases have attracted attention as possible targets for antimicrobial therapy (6). Indeed, several cysteine proteases have been identified as potential targets for such therapy, including the papain-like staphopains A and B from Staphylococcus aureus (7), streptopain (exotoxin B) from Streptococcus pyogenes (3), the gingipains RGP and KGP from Porphyromonas gingivalis (2), PrtH (FDF) from Tannerella forsythia (8), and YopT from Yersinia enterocolitica (9).Recently, we described a novel, heat-resistant protein (Streptomyces papain inhibitor [SPI]) from Streptomyces mobaraensis that inhibited the activity of the cysteine protease papain, as well as (to a lesser extent) the activities of cysteine protease bromelain and the serine protease trypsin in the nanomolar range (10). In the present report, we describe investigations into the inhibitory activities of SPI on bacterial cysteine proteases, i.e., potential virulence factors, and on the growth capabilities of a range of bacterial pathogens. Our results indicate that SPI has the ability to inhibit secreted bacterial cysteine proteases, as well as bacterial growth, and represent a first step in confirming SPI as a potential broadspectrum antibacterial agent.SPI. Preparation of highly purified SPI was performed as described previously (10). Briefly, Streptomyces mobaraensis DSM 40847 T (IPCR 16-22) was cultured at 42°C for 30 h. The heated cell-free super...

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 Km and kcat 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 (Ki 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.