Lysine 219 Participates in NADPH Specificity in a Flavin-Containing Monooxygenase from Saccharomyces cerevisiae

• Published in: Archives of biochemistry and biophysics 1999-12, Vol.372 (2), p.360-366
• Main Authors: Suh, Jung-Keun, Poulsen, Lawrence L, Ziegler, Daniel M, Robertus, Jon D
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Amino Acid Substitution; Binding Sites; Cysteine - metabolism; Escherichia coli - genetics; Flavin-Adenine Dinucleotide - analysis; flavin-containing monooxygenase; Kinetics; Lysine - genetics; Lysine - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; NAD - metabolism; NADP (coenzyme); NADP - metabolism; NADPH recognition; Oxidation-Reduction; oxygen; oxygenases; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - isolation & purification; Oxygenases - metabolism; Phosphates - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequence Alignment; Substrate Specificity
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1530

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O2 to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2′ phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP+ to oxidized yFMO were measured spectroscopically. Kd is 12 μM for the wild-type enzyme and 243 μM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.