Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group.
Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hemoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme.
Cheesman
Matthew J
MJ
Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
Baer
Brian R
BR
Zheng
Yi-Min
YM
Gillam
Elizabeth M J
EM
Rettie
Allan E
AE
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001571:Benzoflavones / Q000378:metabolism; D001671:Biochemistry / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006418:Heme / Q000378:metabolism*; D007850:Lauric Acids / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001571:Benzoflavones; D007850:Lauric Acids; D008024:Ligands; D011994:Recombinant Proteins; C030358:lauric acid; D006418:Heme; C011512:alpha-naphthoflavone; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1