Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases.
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.
Telkov
M V
MV
Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia.
Demina
G R
GR
Voloshin
S A
SA
Salina
E G
EG
Dudik
T V
TV
Stekhanova
T N
TN
Mukamolova
G V
GV
Kazaryan
K A
KA
Goncharenko
A V
AV
Young
M
M
Kaprelyants
A S
AS
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016982:Micrococcus luteus / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D003470:Culture Media; D016207:Cytokines; D011994:Recombinant Proteins; C113704:resuscitation-promoting factor, bacteria; D010447:Peptide Hydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase