Nocardia farcinica nocobactin G (NbtG) is a flavin-dependent monooxygenase that catalyzes the N6-hydroxylation of lysine in the biosynthesis of the siderophore nocobactin. Currently, there are no three-dimensional structures available for N6-lysine monooxygenases. A three-dimensional model was generated from a related enzyme and K64 was selected for mutagenesis to test its potential role in substrate binding.  The resulting K64A mutant was expressed, purified to homogeneity, and the steady-state kinetic parameters determined. For wild-type NbtG, kcat and Km(Lys) values of 12.4 � 0.1 s-1 and 0.20 � 0.01 mM were determined, respectively.  In contrast, the K64A mutant was found to have kcat- and Km(Lys) values of 1.60 � 0.06 s-1 and 4.5 � 0.50 mM, respectively.  The 23-fold decrease in the Km(Lys) value is consistent with K64 being important for lysine binding. This work was supported by a grant from the National Science Foundation MCB-1021384. 

Lys64 is important for substrate binding in the flavoprotein nocobactin G

245th National Meeting (2013)

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