Novel extracellular x-prolyl dipeptidyl-peptidase (DPP) from Streptococcusgordonii FSS2: an emerging subfamily of viridans streptococcal x-prolyl DPPs

Streptococcus gordonii is generally considered a benign inhabitant of the o ral microflora, and yet it is a primary etiological agent in the developmen t of subacute bacterial endocarditis (SBE), an inflammatory state that prop agates thrombus formation and tissue damage on the surface of heart valves. Strain FSS2 produced several extracellular aminopeptidase and fibrinogen-d egrading activities during growth in culture. In this report we describe th e purification, characterization, and cloning of a serine class dipeptidyl- aminopeptidase, an x-prolyl dipeptidyl-peptidase (Sg-xPDPP, for S. gordonii x-prolyl dipeptidyl-peptidase), produced in a pH-controlled batch culture. Purification of this enzyme by anion exchange, gel filtration, and hydroph obic interaction chromatography yielded a protein monomer of approximately 85 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophores is (PAGE) under denaturing conditions. However, under native conditions, th e protein appeared to be a homodimer on the basis of gel filtration and PAG E. Kinetic studies indicated that purified enzyme had a unique and stringen t x-prolyl specificity that is comparable to both the dipeptidyl-peptidase IV/CD26 and lactococcal x-prolyl dipeptidyl-peptidase families. Nested PCR cloning from an S. gordonii library enabled the isolation and sequence anal ysis of the full-length gene. A 759-amino-acid polypeptide with a theoretic al molecular mass of 87,115 Da and a calculated pf of 5.6 was encoded by th is open reading frame. Significant homology was found with the PepX gene fa mily from Lactobacillus and Lactococcus spp. and putative x-prolyl dipeptid yl-peptidases from other streptococcal species. Sg-xPDPP may serve as a cri tical factor for the sustained bacterial growth in vivo and furthermore may aid in the proteolysis of host tissue that is commonly observed during SBE pathology.