Lysine: N6-hydroxylase: stability and interaction with ligands.
Recombinant lysine:N6-hydroxylase, rIucD, which is isolated as an apoenzyme, requires FAD and NADPH for its catalytic function. rIucD preparations have been found to undergo time-dependent loss in monooxygenase function due to aggregation from the initial tetrameric state to a polytetrameric form(s), a process which is reversible by treatment with thiols. Ligand-induced conformational changes in rIucD were assessed by monitoring its CD spectra, DSC profile, and susceptibility to both endo- as well as exopeptidases. The first two methods indicated the absence of any significant conformational change in rIucD, while the last approach revealed that FAD, and its analog ADP, can protect the protein from the deleterious action of proteases. NADPH was partially effective and L-lysine was ineffective in this regard. Deletion of the C-terminal segment, either by treatment with carboxypeptidase Y or by mutagenesis of iucD, results in the loss of rIucD's monooxygenase activity. These findings demonstrate the crucial role of the C-terminal segment in maintaining rIucD in its native conformation.
Dick
S
S
Department of Chemistry, University of Waterloo, Canada.
Marrone
L
L
Duewel
H
H
Beecroft
M
M
McCourt
J
J
Viswanatha
T
T
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism; D043402:Cathepsin A; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D000244:Adenosine Diphosphate; D006899:Mixed Function Oxygenases; C047196:lysine N-epsilon hydroxylase; D002268:Carboxypeptidases; D010450:Endopeptidases; D043402:Cathepsin A; D014357:Trypsin