Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase.
Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).
Joint Center for Structural Genomics (http://www.jcsg.org); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
Qingping
Q
Xu
Joint Center for Structural Genomics (http://www.jcsg.org); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
Hsiu-Ju
HJ
Chiu
Joint Center for Structural Genomics (http://www.jcsg.org); Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Carol L
CL
Farr
Joint Center for Structural Genomics (http://www.jcsg.org); Center for Research in Biological Systems, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Lukasz
L
Jaroszewski
Joint Center for Structural Genomics (http://www.jcsg.org); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Mark W
MW
Knuth
Joint Center for Structural Genomics (http://www.jcsg.org); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
Mitchell D
MD
Miller
Joint Center for Structural Genomics (http://www.jcsg.org); Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Scott A
SA
Lesley
Joint Center for Structural Genomics (http://www.jcsg.org); Center for Research in Biological Systems, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Adam
A
Godzik
Joint Center for Structural Genomics (http://www.jcsg.org); Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Marc-André
MA
Elsliger
Joint Center for Structural Genomics (http://www.jcsg.org); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
Ashley M
AM
Deacon
Joint Center for Structural Genomics (http://www.jcsg.org); Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address: wilson@scripps.edu.
Ian A
IA
Wilson
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D016360:Clostridioides difficile / Q000235:genetics; D018360:Crystallography, X-Ray; D004251:DNA Transposable Elements; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000235:genetics
D004251:DNA Transposable Elements; D006867:Hydrolases
JCSG; Joint Center for Structural Genomics; LT; MAD; MD; MGE; MR; N-acetylglucosamine; N-acetylmuramic acid; NAG; NAM; NIGMS; NIH; National Institute of General Medical Sciences; National Institutes of Health; NlpC/P60 endopeptidase; PSI; Protein Structure Initiative; SSRL; Stanford Synchrotron Radiation Lightsource; TEV; Tn916 family conjugative transposons; asu; asymmetric unit; bacterial lysozyme; bifunctional cell wall lysin; lytic transglycosylase; mobile genetic element; molecular dynamics; molecular replacement; multi-wavelength anomalous dispersion; muramidase; tobacco etch virus