title: Involvement of a Novel Class C Beta-Lactamase in the Transglutaminase Mediated Cross-Linking Cascade of Streptomyces mobaraensis DSM 40847.
abstract: 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.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Zindel
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Ehret
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Ehret
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Hentschel
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Samantha
authors.initials: S
authors.lastname: Witt
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Krämer
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Fiebig
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Jüttner
authors.affiliation: Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Fröls
authors.affiliation: Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Felicitas
authors.initials: F
authors.lastname: Pfeifer
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Darmstadt, Germany.
authors.forename: Hans-Lothar
authors.initials: HL
authors.lastname: Fuchsbauer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D011503:Transglutaminases; D001618:beta-Lactamases