Isolation and crystallization of functionally competent Escherichia coli peptide deformylase forms containing either iron or nickel in the active site.
Three metallo forms of peptide deformylase (PDF, EC 3.5.1.31) of Escherichia coli were prepared and crystallized (space group C2, diffraction limit 1.9 A) for initiating the X-ray structure determination of the metal center in correlation with the catalytic functionality of this enzyme. The native Fe2+ containing enzyme species was directly isolated from overproducing bacteria by using catalase as a buffer additive, which stabilizes the catalytic activity against oxidative destruction. The Ni2+ containing form, which is oxygen-insensitive, was obtained by metal exchange with free Ni2+ and found to be catalytically equally effective (kcat/KM = 10(5) M-1 s-1 for N-formyl-Met-Ala). The Zn2+ form, prepared from the apoenzyme or by displacement of bound Ni2+ by free Zn2+, proved virtually inactive.
Groche
D
D
Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universität, Germany.
Becker
A
A
Schlichting
I
I
Kabsch
W
W
Schultz
S
S
Wagner
A F
AF
D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D002374:Catalase; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D009532:Nickel / Q000737:chemistry; D010084:Oxidation-Reduction; D015032:Zinc / Q000737:chemistry
D009532:Nickel; D007501:Iron; D002374:Catalase; D000581:Amidohydrolases; C020150:formylmethionine deformylase; D015032:Zinc