Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471.
Sphingomyelinase C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a single band of 38 kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF MS was 32,102 Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found at pH 9.0 and 50-55 degrees C, and the enzyme was stable from pH 5 to 10 and up to 37 degrees C. The SMC activity requires Mg(2+) or Mn(2+) and the order of potency to enhance the activity was Zn(2+)> or =Mn(2+)>Cu(2+)> or =Fe(2+). Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were determined as H(2)N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020 bp encoding a signal peptide of 42 amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125 Da. The conserved region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.
Sugimori
Daisuke
D
Department of Industrial System, Fukushima University, Kanayagawa, Japan. sugimori@sss.fukushima-u.ac.jp
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics
D002413:Cations, Divalent; D017931:DNA Primers; D004791:Enzyme Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D004492:Edetic Acid; D013108:Sphingomyelin Phosphodiesterase