Recombinant lysine:N(6)-hydroxylase: effect of cysteine-->alanine replacements on structural integrity and catalytic competence.
Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed.
Dick
Scott
S
Department of Chemistry, University of Waterloo, ON, Canada.
Siemann
Stefan
S
Frey
Harold E
HE
Lepock
James R
JR
Viswanatha
Thammaiah
T
D000409:Alanine / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D006358:Hot Temperature; D007480:Iopanoic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
C121277:1,3-dihydroxypropan-2-one 1,3-diiopanoate; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D004228:Dithionitrobenzoic Acid; D006899:Mixed Function Oxygenases; C047196:lysine N-epsilon hydroxylase; D007480:Iopanoic Acid; D003545:Cysteine; D000409:Alanine