Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N-monooxygenase

Abstract

l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumannii

“…This decrease was mainly due to a reduction in the k cat value. The difference in the k cat value from the oxygen consumption assay and the iodine oxidation assay originated from uncoupling, where hydrogen peroxide is produced and no hydroxylation takes place. , This uncoupling likely takes place due to the structural similarities of cadaverine and putrescine, whereas cadaverine can fit within the active site but is likely not appropriately positioned in comparison to putrescine to the C4a of the isoalloxazine ring on the flavin, resulting in breakdown of the C4a-hydroperoxyflavin intermediate. These results are consistent with putrescine being the preferred substrate for FbsI.…”

Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis

“…Nocardia facinica G, NbtG, is another lysine monooxygenase that has been well-characterized. NbtG displays a 4-fold preference to NADPH and is 70% coupled with NADPH as compared to only 50% with NADH [ 18 , 19 ]. Thus, the ornithine hydroxylases have been shown to have specificity for NADPH, while the lysine monooxygases show lower affinity with NADPH and are less specific, with some preferring NADH (MbsG) and others NADPH (NbtG).…”

Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis

“…Transformed cells were grown in 50 mL of LB media supplemented with 0.1 mg/mL ampicillin overnight. Flasks of 1 L LB media supplemented with ampicillin were inoculated with 8 mL of the pre-growth culture and shaken at 37 °C and 250 rpm until an optical density of 0.6 was obtained. Each flask was induced with 0.1 mM IPTG, the temperature lowered to 18 °C, and the cultures were shaken overnight.…”

“…FMOs are commonly found in natural product biosynthetic pathways, where they play a major role in the addition of functional groups essential for bioactivity. , This family has been categorized into eight classes, A–H, according to structural and mechanistic characteristics . Our group has been studying several members of class B FMOs as these enzymes perform highly specific oxidations useful for biomedical and biotechnological applications. − …”