Novel extracellular x-prolyl dipeptidyl-peptidase (DPP) from Streptococcus gordonii FSS2: an emerging subfamily of viridans Streptococcal x-prolyl DPPs

Abstract

Streptococcus gordonii is generally considered a benign inhabitant of the oral microflora, and yet it is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Strain FSS2 produced several extracellular aminopeptidase and fibrinogen-degrading activities during growth in culture. In this report we describe the 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 hydrophobic interaction chromatography yielded a protein monomer of approximately 85 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. However, under native conditions, the protein appeared to be a homodimer on the basis of gel filtration and PAGE. Kinetic studies indicated that purified enzyme had a unique and stringent 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 analysis of the full-length gene. A 759-amino-acid polypeptide with a theoretical molecular mass of 87,115 Da and a calculated pI of 5.6 was encoded by this open reading frame. Significant homology was found with the PepX gene family from Lactobacillus and Lactococcus spp. and putative x-prolyl dipeptidyl-peptidases from other streptococcal species. Sg-xPDPP may serve as a critical 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.

FIG. 1

SDS-PAGE of fractions from the purification of Sg-x-PDPP and the autoradiography of the purified peptidase. Lane 1, 15 μg of molecular mass markers (phosphorylase B, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa). The following lanes contained boiled, reduced samples: lane 2, 140 μg of 80% ammonium sulfate precipitate; lane 3, 102 μg of eluted peak from DE52 anion exchange; lane 4, 98 μg of peak from Superdex 75 gel filtration wash; lane 5, 55 μg of eluted peak from phenyl Sepharose; lane 6, 39 μg of eluted peak from Mono Q anion exchange; lane 7, 15 μg of CB Sepharose flow-through; lane 8, 6 μg of purified Sg-xPDPP from Gly-Pro Sepharose; lane 9, autoradiograph of [³H]DFP-labeled enzyme exposed for 96 h to X-ray film.

FIG. 2

Multiple sequence alignment of Sg-xPDPP, putative streptococcal PepX genes, and other bacterial homologues. Sequences of Sg-xPDPP (Sgord) deduced from FSS2 genome, putative x-PDPPs from gene products of S. pneumoniae (Spneu) and S. mutans (Smut), cloned x-PDPP from Lactococcus lactis subsp. lactis 763 (Llac763), Lactococcus lactis subsp. cremoris (Lcrem), Lactobacillus delbrueckii subsp. lactis (Ldel17290), and cloned DPPIV from Flavobacterium meningosepticum (Fmenin) and Porphyromonas gingivalis (Pging) were aligned using the ClustalW multiple sequence alignment tool according to homology modeling (grey boxes indicate similarity and black boxes indicate identity). The putative catalytic residues (serine, aspartic acid, and histidine) are denoted with asterisks. The arrow marks a consensus motif flanking the active-site serine in members of the x-PDPP family (PepX). Double lines represent N-terminal and internal fragments used to generate a 965-bp PCR product.