A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis

Abstract

The flavoenzyme nikD is required for the biosynthesis of nikkomycin antibiotics. NikD exhibits an unusual long wavelength absorption band attributed to a charge transfer complex of FAD with an unknown charge transfer donor. NikD crystals contain an endogenous active site ligand. At least four different compounds are detected in nikD extracts, including variable amounts of two ADP derivatives that bind to the enzyme's dinucleotide binding motif in competition with FAD, picolinate (0.07 mol/mol of nikD) and an unknown picolinate-like compound. Picolinate, the product of the physiological catalytic reaction, matches the properties deduced for the active site ligand in nikD crystals. The charge transfer band is eliminated upon mixing nikD with excess picolinate but not by a reversible unfolding procedure that removes the picolinate-like compound, ruling out both compounds as the intrinsic charge transfer donor. Mutation of Trp355 to Phe eliminates the charge transfer band, accompanied by a 30-fold decrease in substrate binding affinity. The results provide definitive evidence for Trp355 as the intrinsic charge transfer donor. The indole ring of Trp355 is coplanar with or perpendicular to the flavin ring in "open" or "closed" crystalline forms of nikD, respectively. Importantly, a coplanar configuration is required for charge transfer interaction. Absorption in the long wavelength region therefore constitutes a valuable probe for monitoring conformational changes in solution that are likely to be important in nikD catalysis.

Figure 1

Stereo views of the active site in the closed (top panel) and open (bottom panel) forms (PDB code 2H2V and 2H2L, respectively). Atoms are colored: C, gold (closed form) or green (open form); O, red; N, blue; S, yellow. For clarity, only selected hydrogen bonds are shown.

Figure 2

Anion exchange HPLC analysis of nikD ligands. Extracts were prepared from nikD isolated from cells grown in TB (panel A) or LB (panel B) medium. Panels C and D are elution profiles obtained with picolinate and AMP, respectively. The chromatograms were developed using method 1. (Compound IV binds tightly to the anion exchange column and is not eluted under these conditions.)

Figure 3

Reversed phase HPLC analysis of nikD ligands. Panel A is the elution profile obtained with an extract prepared from nikD isolated from cells grown in TB medium. Panel B is the elution profile obtained with AMP. The chromatograms were developed using method 3. (Compound III is not eluted under these conditions.)

Figure 4

Effect of phosphodiesterase I treatment on compounds I and IV. HPLC analyses were conducted using a reversed phase column and method 3. Panel A was obtained with an untreated ligand extract from enzyme that had been isolated from cells grown in TB medium. Panel B was obtained after incubating the extract with 0.0095 U/mL of purified phosphodiesterase I for 60 min in 50 mM potassium phosphate buffer, pH 8.8 at 37 °C. Panel C was obtained with AMP.

Figure 5

Effect of unfolding and renaturation on the spectral properties and the endogenous ligand content of nikD. Panel A shows absorption spectra of native (solid black line) and refolded (dashed red line) nikD in 100 mM potassium phosphate buffer, pH 8.0, at 22 °C. Absorption spectra for the corresponding heat extracts are shown in panel B. Spectra are normalized to the same enzyme concentration. The inset in panel A shows previously reported (5) absorption spectra of free nikD (solid line) and the enzyme·picolinate complex (dashed line) obtained at 25 °C in 100 mM potasium phosphate buffer, pH 8.0, containing 0 and 2.27 mM picolinate, respectively.

Figure 6

Comparison of the spectral properties of the Trp355Phe mutant with wild type nikD. Spectra for the mutant (solid line) and wild type (dashed line) enzyme were recorded in 100 mM potassium phosphate buffer, pH 8.0 at 22 °C.

Scheme 1

Role of nikD in the biosynthesis of the peptidyl moiety of nikkomycins.

Scheme 2

Postulated two-step mechanism for formation of a redox-active enzyme·substrate complex.