New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

Abstract

Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

Fig. 1. Structure of STM3605

(a). Superposition of molecule A (purple) and B (pink). (b). Covalent dimer created through two disulfide bonds shown in 2mFo-DFc electron density map contoured at 1σ level. (c). Dimethylated lysine residue (Lys26) shown in 2mFo-DFc electron density map contoured at 1σ level.

Fig. 2. Comparison between STM3605t and T4 lysozyme

(a). Superposition of STM3605t (molecule A, purple) with T4 lysozyme (grey, PDB code 151L). H and S labels refer to T4 lysozyme, while α and β correspond to STM3605t. (b). Superposition of the STM3605t noncovalent dimer (molecule A – purple, molecule B’ – pink) with T4 lysozyme. (c). Superposition of T4 lysozyme (grey surface) with STM3605t (purple) showing the C-terminal tail (in 2mFo-DFc electron density map contoured at 1σ level) of STM3605 going through the enzyme active site.

Fig. 3. HPLC size exclusion chromatography of native STM3605 protein and its mutants

WT SMT3605f is shown as yellow circle, double mutant Q20E/V35T is blue and single mutant Q20E is light blue, standards (pink circles) are 1. aprotinin (6.5 kDa), 2. ribonuclease A (13.7 kDa), 3. carbonic anhydrase (29 kDa), 4. ovalbumin (43 kDa), 5. conalbumin (75 kDa), 6. aldolase (158 kDa) and 7. thyroglobulin (669 kDa).

Fig. 4. Comparison of the putative “active site” of STM3605 with T4 lysozyme

(a). Superposition of the STM3605t (purple and pink) with T4 lysozyme (grey, PDB code 151L) with residues important for catalysis in T4 lysozyme and their STM3605 counterparts (labeled) shown in a stick representation. (b). The same as in A with active site cavities calculated in SURFNET [43] shown as purple surface (STM3605, calculated for a protein with residues Gly95 – Ile103 excluded) and grey mesh (T4 lysozyme).

Fig. 5. Cell lysis assay with M. luteus ATCC No. 4698 strain of native STM3605 protein and its mutants

WT SMT3605f is shown as red circles, single mutant Q20E is green, and double mutant Q20E/V35T is blue. Yellow circles correspond to the control with no protein added.

Fig. 6. Micelle assay of native STM3605 protein and its mutants

Lines 1-3 are controls with photoreaction center proteins (3 bands) - 1. Control, 2. detergent phase, 3. water phase. Lines 4-6 are for HEWL lysozyme - 4. Lysozyme control, 5. detergent phase, 6. water phase, lines 7-9 are for WT STM3605 - 7. WT STM3605 control, 8. detergent phase, 9. water phase, lines 10-12 are for STM3605 Q20E single mutant – 10. Q20E control, 11. detergent phase, 12. water phase and lines 13-15 are for STM3605 Q20E/V35T double mutant – 13. Q20E/V35T control, 14. detergent phase, 15. water phase.

Fig. 7

Multiple sequence alignment of STM3605 and its homologs: the other two lysozyme-like proteins in S. Typhimurium as well as T4 and P22 phage lysozymes. Sequence identity between STM3605 and the other sequences in this alignment is less than 30%.