Structural and functional characterization of NikO, an enolpyruvyl transferase essential in nikkomycin biosynthesis

Abstract

Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, only little is known about their complex biosynthesis. The genes encoding for proteins involved in the biosynthesis of the nucleoside moiety of nikkomycins are co-transcribed in the same operon, comprising the genes nikIJKLMNO. The gene product NikO was shown to belong to the family of enolpyruvyl transferases and to catalyze the transfer of an enolpyruvyl moiety from phosphoenolpyruvate to the 3'-hydroxyl group of UMP. Here, we report activity and inhibition studies of the wild-type enzyme and the variants C130A and D342A. The x-ray crystal structure revealed differences between NikO and its homologs. Furthermore, our studies led to conclusions concerning substrate binding and preference as well as to conclusions about inhibition/alkylation by the antibiotic fosfomycin.

SCHEME 1.

The reaction catalyzed by NikO, the structure of the synthesized compound, aminohexuronic acid, and the two most abundantly found nikkomycins are shown. The enolpyruvyl group is transferred onto 3′-OH of UMP.

FIGURE 1.

Crystal structure of NikO. A, the final refined NikO structure (comprising two molecules in the asymmetric unit) is shown in a graphic representation. The molecules exhibit a rarely found inverse α,β-barrel fold and are colored according to their secondary structure elements (α-helices, light blue or light green; β-strands, dark blue or dark green; loops, gray). B, modeled loop structure of residues 127–139 in NikO. The best scoring loop model was chosen, exhibiting an open loop conformation. The cysteine residue (Cys-130) of this loop is shown as sticks. C, active site of NikO with catalytic residues shown as sticks. NikO is colored in dark green, and MurA is in blue. Active site residues of NikO are colored yellow. Residues are labeled according to NikO numbering. D, structure of the loop region (residues 180–192) in NikO. The structure is shown in a graphic representation with the loop colored in yellow. The figure was prepared with PyMOL (49).

FIGURE 2.

Close-up view of the lowest energy binding mode obtained upon docking of EPUMP into the active site of NikO (EPUMP, white; NikO-residues, green). The figure was prepared with PyMOL (49).

FIGURE 3.

Michaelis-Menten plots for UMP (upper panel) and PEP (lower panel).

FIGURE 4.

Fosfomycin binding. A, NikO binds to fosfomycin in the absence of UMP. Upper panel, gel stained with Coomassie Blue; lower panel, gel stained with ProQ Diamond. The lanes were loaded with the following samples: lane 1, MurA; lane 2, MurA and UDPNAG; lane 3, MurA, UDPNAG and fosfomycin; lane 4, NikO; lane 5, NikO and fosfomycin; lane 6, protein marker. B, binding of fosfomycin to NikO determined by isothermal titration microcalorimetry. 346 μm NikO was titrated with 5 mm fosfomycin.

FIGURE 5.

The C130A variant of NikO does not bind fosfomycin. A gel stained with ProQ Diamond is shown. The lanes were loaded with the following samples: lane 1, NikO C130A and fosfomycin; lane 2, NikO C130A; lane 3, NikO and fosfomycin; lane 4, NikO; lane 5, protein marker. +ffm, fosfomycin added; −ffm, no fosfomycin added; MW-Std., molecular weight standard.

FIGURE 6.

Determination of the limiting rate of k_inact. In the figure, the reciprocal value of k_inact was plotted against the reciprocal value of the fosfomycin concentration. The y-intercept is 12.8 min, so the limiting rate of k_inact was determined to be 0.08 min⁻¹. To determine k_inact, time-dependent inhibition of NikO by fosfomycin was measured, and the residual enzymatic activity was plotted against the incubation time. For each concentration of fosfomycin in the incubation mix (50 μm, 100 μm, 250 μm, 500 μm, 1 mm, respectively), k_inact was determined by fitting the data to the equation y = y0 + exp(−k_inact × x).