Regulation of a phage endolysin by disulfide caging.

In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, Lyz(P1), of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz(103), the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz(103) does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz(103) is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, Lyz(P1) and Lyz(103) define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz(103) is the first enzyme found to be regulated by disulfide bond caging of its active site.

Regulation of a muralytic enzyme by dynamic membrane topology.

R(21), the lysozyme of coliphage 21, has an N-terminal signal-anchor-release (SAR) domain that directs its secretion in a membrane-tethered, inactive form and then its release and activation in the periplasm. Both genetic and crystallographic studies show that the SAR domain, once extracted from the bilayer, refolds into the body of the enzyme and effects muralytic activation by repositioning one residue of the canonical lysozyme catalytic triad.

Annotating enzymes of unknown function: N-formimino-L-glutamate deiminase is a member of the amidohydrolase superfamily.

The functional assignment of enzymes that catalyze unknown chemical transformations is a difficult problem. The protein Pa5106 from Pseudomonas aeruginosa has been identified as a member of the amidohydrolase superfamily by a comprehensive amino acid sequence comparison with structurally authenticated members of this superfamily. The function of Pa5106 has been annotated as a probablechlorohydrolase or cytosine deaminase. A close examination of the genomic content of P. aeruginosa reveals that the gene for this protein is in close proximity to genes included in the histidine degradation pathway. The first three steps for the degradation of histidine include the action of HutH, HutU, and HutI to convert L-histidine to N-formimino-L-glutamate. The degradation of N-formimino-L-glutamate to L-glutamate can occur by three different pathways. Three proteins in P. aeruginosa have been identified that catalyze two of the three possible pathways for the degradation of N-formimino-L-glutamate. The protein Pa5106 was shown to catalyze the deimination of N-formimino-L-glutamate to ammonia and N-formyl-L-glutamate, while Pa5091 catalyzed the hydrolysis of N-formyl-L-glutamate to formate and L-glutamate. The protein Pa3175 is dislocated from the hut operon and was shown to catalyze the hydrolysis of N-formimino-L-glutamate to formamide and L-glutamate. The reason for the coexistence of two alternative pathways for the degradation of N-formimino-L-glutamate in P. aeruginosa is unknown.