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 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 to monitor conformational changes in solution that are likely to be important in nikD catalysis.Nikkomycins are peptidyl nucleoside antibiotics that block the biosynthesis of chitin by inhibiting chitin synthase (1). Chitin, the second most abundant polysaccharide in nature, maintains the structural integrity of the cell wall in fungi and the exoskeleton of insects and other invertebrates. Neither chitin nor chitin synthase is found in mammals. Nikkomycins are effective for the therapeutic treatment of fungal infections in humans and as easily degraded insecticides in agriculture (2).Biosynthesis of the nikkomycin peptide occurs via a nonribosomal pathway. The first step is catalyzed by an aminotransferase that converts L-lysine to an α-keto intermediate that cyclizes and dehydrates to yield piperideine-2-carboxylate (P2C), a compound that can exist in imine and enamine forms (3). The second step is catalyzed by nikD in a reaction that involves a remarkable 4-electron oxidation of P2C to picolinate, accompanied by reduction of 2 mol of oxygen to hydrogen peroxide (Scheme 1) (4-6). NikD contains 1 mol of covalently bound FAD (8″-S-cysteinyl-FAD), exists as a monomer in solution and acts as an obligate 2-electron acceptor. The initial 2-electron oxidation of P2C to dihydropicolinate (DHP) is rate- † This work was supported in part by Grant AI 55590 (M. S. J.) from the National Institutes of Health.*To whom correspondence should be addressed. Phone: (215) NikD exhibits two absorption maxima in the visible region, a feature characteristic of flavincontaining enzymes. However, the enzyme also ...

Probing the Role of Active Site Residues in NikD, an Unusual Amino Acid Oxidase That Catalyzes an Aromatization Reaction Important in Nikkomycin Biosynthesis

“…Mutation of Trp355 to Phe eliminates the long-wavelength absorption band observed with wild-type nikD at slightly alkaline pH (17). Significantly, titration of Trp355Phe from pH 8.0 to 10.4 results in spectral changes that are fully characteristic of FAD ionization at N(3)H (pK a = 9.15 ± 0.05) (Figure 4A, inset).…”

“…Interestingly, the crystal structures of the open and closed forms of the picolinate complex with wild-type nikD led us to propose a two-step binding mechanism. In this model, ligands were thought to form an initial open complex with the indole ring of Trp355 parallel to the flavin ring, followed by displacement of Trp355 to produce a more stable complex with ligand stacked above the flavin ring (17). However, the observed rate of complex formation with wild-type enzyme was found to exhibit a linear dependence on picolinate concentration, consistent with a simple one-step approach to equilibrium (9).…”

“…PCR reactions were conducted using a Hybaid Touchdown Thermocycler or a Perkin Elmer 9600 GeneAmp PCR System and products were purified as previously described (17). The left-hand fragment was generated using START (external primer) as forward primer and an internal backward primer containing the desired mutation (see Table 1).…”

“…Solutions of ligand-free nikD at weakly alkaline pH exhibit two typical flavin absorption maxima at λ > 300 nm plus an unusual long-wavelength absorption band due to charge-transfer interaction between FAD and Trp355 (17). The charge-transfer interaction requires a coplanar configuration of the flavin and indole rings, as observed in the crystal structure of the open form of the enzyme•picolinate complex.…”

ADP Competes with FAD Binding in Putrescine Oxidase

“…The binding of ADP in an FAD binding domain is not unique for PuO Rh . In NikD, a flavoprotein involved in nikkomycin biosynthesis, significant amounts of ADP derivatives were found to be present in the isolated protein, instead of FAD (20). Binding of ADP leads to an inactive enzyme, as it replaces the FAD redox cofactor.…”