A 46-kDa hemolytic
protein, referred to as
cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa
proteins were purified to homogeneity. Both
proteins expressed identical
biological and functional characteristics. In addition to its
biological function of lysing erythrocytes and hemoxidizing the
hemoglobin to
methemoglobin,
cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g.,
cysteine) producing H2S, NH3, and
pyruvate. This
cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(
cat) = 12 s(-1).
Cystathionine and S-aminoethyl-
L-cysteine were also substrates for the
protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3,
pyruvate,
homocysteine (from
cystathionine), and
cysteamine (from S-aminoethyl-
L-cysteine). The
enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-
mercaptoethanol. It was not inhibited by the
proteinase inhibitor
TLCK (N alpha-p-tosyl-
L-lysine chloromethyl
ketone),
pronase, or
proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the
polypeptide. We hypothesize that
cystalysin is a pyridoxal-5-phosphate-containing
enzyme, with activity of an alphaC-N and betaC-S
lyase (
cystathionase) type. Since large amounts of H2S have been reported in deep
periodontal pockets,
cystalysin may also function in vivo as an important virulence molecule.