A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a pre-organized binding site for superoxide anion, an obligatory intermediate in the 2-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. Firstly, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX•chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analog (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX•chloride complex and a ternary MSOX•chloride•MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule, and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1 and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride: (i) acts as an oxygen surrogate; (ii) is an effective probe of polar oxygen activation sites; (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

NikD catalyzes a remarkable aromatization reaction that converts piperideine-2-carboxylate (P2C) to picolinate, a key component of the nonribosomal peptide in nikkomycin antibiotics. The enzyme exhibits a FAD-Trp355 charge-transfer band at weakly alkaline pH that is abolished upon protonation of an unknown ionizable residue that exhibits a pKa of 7.3. Stopped-flow studies of the reductive half-reaction with wild-type nikD and P2C show that the enzyme oxidizes the enamine tautomer of P2C but do not distinguish among several possible paths for the initial 2-electron oxidation step. Replacement of Glu101 or Asp276 by a neutral residue does not eliminate the ionizable group, although the observed pKa is 1 or 2 pH units higher, respectively, compared with wild-type nikD. Importantly, the mutations cause only a modest decrease (< 5-fold) in the observed rate of oxidation of P2C to dihydropicolinate. The results rule out the only possible candidates for a catalytic base in the initial 2-electron oxidation step. This outcome provides compelling evidence that nikD oxidizes the bond between N(1) and C(6) in the enamine tautomer of P2C, ruling out alternative paths that require an active site base to mediate the oxidation of a carbon-carbon bond. Because the same restraint applies to the second 2-electron oxidation step, the dihydropicolinate intermediate must be converted to an isomer that contains an oxidizable carbon-nitrogen bond. A novel role is proposed for reduced FAD as an acid-base catalyst in the isomerization of dihydropicolinate.

NikD is a flavoprotein oxidase that catalyzes the oxidation of piperideine-2-carboxylate (P2C) to picolinate in a remarkable aromatization reaction comprising two redox cycles and at least one isomerization step. Tyr258 forms part of an "aromatic cage" that surrounds the ring in picolinate and its precursors. Mutation of Tyr258 to Phe does not perturb the structure of nikD but does affect the coupling of the two redox cycles and causes a 10-fold decrease in turnover rate. Tyr258Phe catalyzes a quantitative 2-electron oxidation of P2C but only 60% of the resulting dihydropicolinate intermediate undergoes a second redox cycle to produce picolinate. The mutation does not affect product yield with an alternate substrate (3,4-dehydro-L-proline) that is aromatized in a single 2-electron oxidation step. Wild-type and mutant enzyme exhibit identical rate constants for P2C oxidation to dihydropicolinate and isomerization of a reduced enzyme•dihydropicolinate complex. The observed rates are 200-and 10-fold faster, respectively, than the mutant turnover rate. Picolinate release from Tyr258Phe is 100-fold faster than turnover. The presence of bound substrate or product is a key factor in oxygen activation by wild-type nikD, as judged by the 10-to 75-fold faster rates observed for complexes of the reduced enzyme with picolinate, benzoate or 1-cyclohexenoate, a 1-deaza P2C analog. The reduced Tyr258Phe•1-cyclohexenoate complex is 25-fold less reactive with oxygen than the wild-type complex. We postulate that mutation of Tyr258 causes subtle changes in active site dynamics that promote release of the reactive dihydropicolinate intermediate and disrupt the efficient synchronization of oxygen activation observed with wild-type nikD.NikD, an unusual flavoprotein oxidase, catalyzes a remarkable aromatization reaction involving two redox cycles and plays a critical role in the biosynthesis of nikkomycin antibiotics. Nikkomycins are potent antifungal agents that block cell wall formation by inhibiting the biosynthesis of chitin, the second most abundant polysaccharide in nature (2,3). Nikkomycins contain a nonribosomal peptide linked by a peptide bond to a nucleoside moiety. † This work was supported in part by Grant AI 55590 (M. S. J.) from the National Institutes of Health. ‡ The atomic coordinates for the crystal structure of the Tyr258Phe mutant form of nikD can be accessed through the RCSB Protein Data Bank under accession number 3HZL.

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