title: Structural design of the active site for covalent attachment of the heme to the protein matrix: studies on a thermostable cytochrome P450.
abstract: The molecular basis of the post-translational modification involving covalent attachment of the heme with a glutamic acid observed in some enzymes of the CYP4 family of heme monooxygenases has been investigated using site-directed mutagenesis of CYP175A1 from Thermus thermophilus. Earlier studies of CYP4 as well as the G248E mutant of CYP101A1 showed covalent linkage of the heme to a conserved glutamic acid of helix I. We have introduced Glu/Asp at the Leu80 position in the β-turn of CYP175A1, on the basis of molecular modeling studies, to assess whether formation of such a covalent linkage is limited only to helix I or whether such modification may also take place with the residue that is spatially located at a position appropriate for activation by the heme peroxidase reaction. Tandem mass spectrometry analyses of the tryptic digest of the wild type and mutants of CYP175A1 were conducted to identify any heme-bound peptide. Tryptic digestion of the L80E mutant of CYP175A1 preincubated with H(2)O(2) showed formation of GLE(-heme)TDWGESWKEARK supporting covalent linkage of Glu80 with the heme in the mutant enzyme. No such heme-bound peptides were found if the sample was not preincubated in H(2)O(2), indicating no activation of the Glu by the heme peroxidase reaction, as proposed earlier. The wild type or L80D mutant of the enzyme did not give any heme-bound peptide. Thus, the results support the idea that covalent attachment of the heme to an amino acid in the protein matrix depends on the structural design of the active site.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Navy Nagar, Colaba, Mumbai 400 005, India.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Goyal
authors.forename: Megha S
authors.initials: MS
authors.lastname: Deshpande
authors.forename: Shyamalava
authors.initials: S
authors.lastname: Mazumdar
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000378:metabolism
chemical_list: D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide