Proteins of the Rpf (Resuscitation Promoting Factor) Family Are Peptidoglycan Hydrolases

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M. V. Telkov¹, G. R. Demina¹, S. A. Voloshin¹, E. G. Salina¹, T. V. Dudik¹, T. N. Stekhanova¹, G. V. Mukamolova^1,2, K. A. Kazaryan¹, A. V. Goncharenko¹, M. Young², and A. S. Kaprelyants^1*

¹Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia; fax: (495) 954-2732; E-mail: arseny@inbi.ras.ru

²Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK; fax: +44 (1970) 622-354; E-mail: miy@aber.ac.uk

^* To whom correspondence should be addressed.

Received July 22, 2005; Revision received September 27, 2005

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The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant “non-culturable” M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N´,N´´-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N´,N´´-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

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KEY WORDS: proteins of the Rpf family, peptidoglycan hydrolases, “non-culturable” cells

DOI: 10.1134/S0006297906040092

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