Characterization of L-cysteine desulfhydrase from Prevotella intermedia.
BACKGROUND
Hydrogen sulfide is responsible for lysis of red blood cells and is a major compound for oral malodor. To clarify the production mechanism of hydrogen sulfide in Prevotella intermedia, we found an L-cysteine desulfhydrase gene (lcs) homologue on the genome database of P. intermedia ATCC25611 and characterized its gene product.


METHODS
The lcs gene homologue cloned into pGEX6p-1 vector was expressed in Escherichia coli and purified. Lcs activity was assayed by detection of the reaction products (hydrogen sulfide and pyruvate) or its derivatives from L-cysteine. Site-directed mutagenesis was used to convert an amino acid of the Lcs molecule.


RESULTS
The purified lcs gene product catalysed the degradation of L-cysteine to pyruvate, ammonia, and hydrogen sulfide, indicating that the protein is L-cysteine desulfhydrase. The enzyme required pyridoxal 5'-phosphate as a cofactor, and it was highly active at pH 7.0 and completely inhibited by ZnCl(2). The K(m) and V(max) of the enzyme were 0.7 mm and 4.2 micromol/min/mg, respectively. Replacement of Tyr-59, Tyr-118, Asp-198, and Lys-233 with any of the amino acids resulted in the complete disappearance of Lcs activity, implying that these amino acids are essential for enzyme activity. In addition, hydrogen sulfide produced by this enzyme lysed sheep red blood cells and modified hemoglobin.


CONCLUSIONS
These results show the enzymatic properties of L-cysteine desulfhydrase from P. intermedia ATCC25611 and also suggest that the Lcs enzyme, which produces hydrogen sulfide from L-cysteine, is closely associated with the pathogenesis of P. intermedia.
Yano
T
T
Department of Periodontology, Showa University School of Dentistry, Shinagawa-ku, Tokyo, Japan.
Fukamachi
H
H
Yamamoto
M
M
Igarashi
T
T
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006461:Hemolysis / Q000235:genetics* / Q000502:physiology; D006862:Hydrogen Sulfide / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D018713:Prevotella intermedia / Q000201:enzymology*; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D003542:Cystathionine gamma-Lyase; D006862:Hydrogen Sulfide