The biosynthetic incorporation of selenomethionine and telluromethionine into pyrrolidone carboxyl peptidase (PYRase) from S. aureus.
Heavy-atom derivatives of PYRase proteins prepared in the past have been unsuitable for x-ray diffraction analysis. Thus, we propose utilizing unnatural metalloid-containing amino acids as an alternative to heavy-atom derivatization. Selenomethionine-containing proteins analyzed by multiwavelength anomalous diffraction provides a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray Crystallography [Hendrickson, et al., 1991 and references therein]. Telluromethionine-containing proteins offer the same investigational potential, and additionally allow further simplification of the data collection technique by requiring only traditional methods of phase analysis [Boles et al., 1995 and references therein]. We sought to introduce the required Se and Te atoms into Staphylococcus aureus Pyrrolidone Carboxyl Peptidase (PYRase) via selenomethionine (SeMet) and telluromethionine (TeMet). Complete incorporation of SeMet into S. aureus PYRase was succeeded with little change in enzymatic properties. Incomplete incorporation (75%) of TeMet was accomplished in preparing TeMet-PYRase, however, representing the highest incorporation to date of a tellurium-containing amino acid. Enzymatic properties remained unchanged when TeMet was incorporated. We report herein the biosynthetic substitution and expression, protein purification and comparative biochemistry of SeMet-PYRase and TeMet-PYRase.
Boles
J O
JO
Department of Chemistry, Tennessee Technological University Cookeville 38505, USA.
Yu
H N
HN
Patti
J M
JM
D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D015394:Molecular Structure; D011751:Pyroglutamyl-Peptidase I / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D012645:Selenomethionine / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013691:Tellurium / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins; C094617:telluromethionine; D012645:Selenomethionine; D008715:Methionine; D011751:Pyroglutamyl-Peptidase I; D013691:Tellurium