AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase

Abstract

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple‐drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β‐fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn‐type or DAP‐type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature T m 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29

“…Advances in research on thermophilic bacteriophages and their proteins have opened new research avenues their applications at elevated temperatures, e.g., in food processing conditions, including pasteurization. A limited number of thermostable endolysins has been described in the literature, but their number grows constantly and this work fits well into this trend (Matsushita and Yanase, 2008;Ye and Zhang, 2008;Plotka et al, 2014Plotka et al, , 2015Choi and Kong, 2023;Jasilionis et al, 2023). Here, we characterize the PhiKo endolysin derived from extremophilic T. thermophilus HB27 bacteriophage phiKo.…”

“…The Ts2631 endolysin that shows 49.0% primary sequence identity to PhiKo endolysin has T m = 99.82°C (Plotka et al, 2015), and the Ph2119 endolysin (52.4% primary sequence identity with PhiKo) has T m = 103.56°C (unpublished results). The melting temperature of another thermostable endolysin AmiP of Thermus parvatiensis prophage is 102.6°C (Jasilionis et al, 2023). As can be observed, the melting temperature of all mentioned endolysins differs only by a few degrees and is in a range of 91.70°C-103.56°C.…”