The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity.
dsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C(4)-C(18)), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C(16) and C(18)). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca(2+) and Mn(2+). To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.
Gil
Filipa
F
Unidade dos Retrovirus e Infecções Associadas, Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Portugal.
Catalão
Maria João
MJ
Unidade dos Retrovirus e Infecções Associadas, Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Portugal.
Moniz-Pereira
José
J
Unidade dos Retrovirus e Infecções Associadas, Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Portugal.
Leandro
Paula
P
Unidade de Biologia Molecular e Biopatologia Experimental, iMed, Faculty of Pharmacy, University of Lisbon, Portugal.
McNeil
Michael
M
Department of Microbiology Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Pimentel
Madalena
M
Unidade dos Retrovirus e Infecções Associadas, Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Portugal.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002087:Butyrates / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D003067:Coenzymes / Q000494:pharmacology; D017124:Conserved Sequence; D010450:Endopeptidases / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D002087:Butyrates; D002413:Cations, Divalent; D003067:Coenzymes; D008670:Metals; D011994:Recombinant Proteins; D014764:Viral Proteins; C033592:4-nitrophenyl butyrate; D008049:Lipase; D010450:Endopeptidases; C001143:endolysin