Structure and function of a cyanophage-encoded peptide deformylase

Abstract

Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.

Figure 1

Global distribution of phage-encoded PDFs. GOS-sampling locations (blue dots) and relative abundances of phage-encoded PDFs (white circles) in the GOS metagenomes are plotted on a global map of marine chlorophyll concentrations. See Supplementary Table S1 for the data plotted.

Figure 2

(a) Inhibition of the Synechococcus phage S-SSM7 PDF (blue) and the PDF from Synechococcus elongatus PCC 6301 (red) by actinonin. Enzyme: 0.2 nℳ PDF. Substrate: 1.5 mℳ fMLIS, the N-terminal tetrapeptide of the D1 protein encoded by the phage. (b) Kinetic assays comparing the activity of the Synechococcus phage S-SSM7 PDF (blue) with that from S. elongatus PCC 6301 (red) on three different N-terminal tetrapeptide substrates derived from phage or bacterial D1 proteins (see Table 1). Red square=phage D1, fMLIS; solid red circle=bacterial D1, fMTSI; open red circle=bacterial D1, fMTTA.

Figure 3

Comparison of the Synechococcus phage S-SSM7 PDF amino-acid sequence with that of other PDFs. (a) Multiple sequence alignment of selected PDFs showing the three conserved motifs and the C-terminal domain. Included are PDFs from: A. thaliana mitochondria; A. thaliana chloroplasts; Synechococcus phage S-SSM7; Synechococcus elongatus PCC 6301; and P. falciparum apicoplast. Highlighted regions are the PDF-specific G-motif (GΦGΦAAXQ), C-motif (EGCXS) and H-motif (QHEXDHLXG), as well as the variable C-terminal domain (where Φ=any hydrophobic amino acid and X=any amino acid). Degree of conservation of amino acids at each position: *=absolutely conserved; :=different but very similar amino acids; .=different but somewhat similar amino acids; blank=dissimilar amino acids or gaps. (b) A phylogenetic tree of 51 PDF proteins from bacteria, phage and eukaryote organelles.

Figure 4

(a) Crystal structure of Synechococcus phage S-SSM7 peptide deformylase (PDF). (b) Overlay of phage S-SSM7 PDF (green), Synechococcus elongatus PCC 6301 PDF (cyan) and A. thaliana chloroplast PDF1B (magenta) shows striking similarity of protein folds, as well as the position of the zinc ion in the active site. (c) Evaluation of the active site residues around the zinc ion reveals strong conservation among these three PDFs: phage S-SSM7 (green), Synechococcus (cyan) and chloroplast (magenta). (d) Comparison of the residues at the entry to the active site in the phage (green), Synechococcus (cyan) and chloroplast (magenta) PDFs. Green sticks=phage asparagine 99; cyan sticks=Synechococcus tyrosine 116; magenta sticks=chloroplast tyrosine 178.

Figure 5

(a) Crystal structure of Synechococcus phage S-SSM7 PDF binding the inhibitor actinonin. (b) Interaction of actinonin with the active site residues of the phage PDF showing polar interactions. Residues from the C- and G-motifs are shown as green sticks, actinonin as orange sticks.