Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis

Abstract

N-hydroxylating monooxygenases (NMOs) are a subclass of flavindependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in some Streptomyces sp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (k cat , 3.0 ± 0.01 s −1 ) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higher k cat /K M value for NADPH compared to NADH and has a narrow optimal pH range (7.5−8.0). Contrary to other NMOs where the k red is rate-limiting, FzmM exhibits a very fast k red (50 ± 0.01 s −1 at 4 °C) with NADPH. NADPH binds at a K D value of ∼400 μM, and hydride transfer occurs with pro-R stereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than the k cat . This reaction is enhanced in the presence of Asp with a slightly faster k ox than the k cat , suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs. 65 characterization of the nitro-forming NMO FzmM providing a 66 detailed report of its mechanism toward the formation of 67 nitrosuccinate.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center

“…Overall, these kinetics and spectral properties closely resemble those originally described for FzmM from Streptomyces sp. XY332 ( 23 ).…”

“…These initial findings have been followed by an increasing interest in NMOs and many enzymes belonging to this family have been discovered in the past years ( 19 , 20 ). A subgroup of NMOs that are attracting considerable interest are PcXL, FzmM, and its homolog CreE ( 21 , 22 , 23 , 24 , 25 , 26 ). They were isolated from Streptomyces species and reported to participate in the biosynthesis of phosphonocystoximate, fosfazinomycin, and cremeomycin, respectively.…”

Kinetic Characterization and Identification of Key Active Site Residues of the L‐Aspartate N‐Hydroxylase, CreE

“…A k cat of 4.28 � 0.07 s À 1 and a K M of 0.82 � 0.06 mM were measured with L-aspartate, which are similar to previously reported values for homologous enzymes (Figure 1 & Table 1). [13,17] We observed little change on the k cat value with D-aspartate but noted a ~23-fold increase in the K M value, suggesting that CreE preferentially binds Laspartate (Figure 1 & Table 1). A previous report showed that SV2À NMO was not active with D-aspartate by monitoring the rate of NADPH oxidation.…”

“…[11,16] However, CreE is an unusual member of this subclass, because the enzyme catalyzes three sequential hydroxylation reactions of L-aspartate to yield nitrosuccinate, in contrast to the typical single oxidation reaction of NMOs (Scheme 1B). [8,11,13,17] Here we present the kinetic characterization of CreE. Steady-state kinetic studies demonstrated that CreE is stereospecific for L-aspartate and that the oxidation is highly coupled.…”

“…Oxygen was removed from the enzyme sample, buffer, and substrate stock as described previously. [17,23] NADPH was transferred directly to the chamber and resuspended in the anaerobic activity buffer (100 mM potassium phosphate, pH 7.5, 100 mM NaCl). For all stopped-flow experiments, the concentrations listed are after mixing.…”