The Structure and Function of Modular Escherichia coli O157:H7

16 Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial 17 candidates to combat antimicrobial resistance. Considering the catalytic and structural 18 diversity of endolysins, there is a paucity of structural data to inform how these enzymes 19 work at the molecular level—key data that is needed to realize the potential of endolysin20 based antibacterial agents. Here, we determine the atomic structure and define the enzymatic 21 function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic 22 analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative 23 endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The 24 crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains 25 connected by a linker region but packed together. LysT84 was determined to be a monomer 26 in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed 27 that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a 28 multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular 29 protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues 30 positioned on either side of the active site cavity; mutagenesis demonstrating these residues 31 are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the 32 enzymatically active domain is dynamic, allowing the appropriate positioning of these 33 catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural 34 basis for peptidoglycan degradation by LysT84 which supports rational engineering of 35 related endolysins into effective antibacterial agents. 36 37 D ow naded rom http://pndpress.com /bchem j/article-oi/10.1042/BC J20210701/9/bcj-2021-0701.pdf by gest on 24 D ecem er 2021 Bchem al Jornal. This is an Acepted M ancript. ou re encuraged to se he Vrsion of R eord tat, w en puished, w ill relace his vesion. he m st up-tote-version is avilable at https://drg/10.1042/BC J210701