Factors That Affect Oxygen Activation and Coupling of the Two Redox Cycles in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase

Abstract

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.

Role of Active Site Histidines in the Two Half-Reactions of the Aryl-Alcohol Oxidase Catalytic Cycle

“…The mechanism of O 2 reduction by AAO (flavin hydroquinone form) with formation of two products (flavin quinone and H 2 O 2 ) and one possible intermediate (C4a-hydroperoxide) was investigated using a combination of experimental and computational methods. A possible intermediate enzyme species was detected by global analysis of the spectral changes during stopped-flow reoxidation of AAO (species B in Figure ), a similar two-step reoxidation model has been reported for cholesterol oxidase and some other flavooxidases. , However, it does not show the characteristic spectrum of the flavin C4a-hydroperoxide . Computational modeling supports this conclusion.…”

“…A possible intermediate enzyme species was detected by global analysis of the spectral changes during stopped-flow reoxidation of AAO (species B in Figure 4), a similar two-step reoxidation model has been reported for cholesterol oxidase and some other flavooxidases. 49,50 However, it does not show the characteristic spectrum of the flavin C4a-hydroperoxide. 51 Computational modeling supports this conclusion.…”

Pleiotropic Impact of a Single Lysine Mutation on Biosynthesis of and Catalysis by N-Methyltryptophan Oxidase

“…However, an accelerated rate of oxygen reduction is observed with reduced nikD 3 ligand complexes. 15 The results suggest that Lys259 may play a role in oxygen activation by MTOX. However, a very different scenario is also possible, as judged by results obtained for a highly conserved histidine residue in members of the glucose-methanol-choline oxidase family.…”

“…In fact, the reaction of free reduced nikD with oxygen is very slow, similar that observed for the corresponding reaction with free flavin. However, an accelerated rate of oxygen reduction is observed with reduced nikD•ligand complexes (15). …”

Characterization of a Protein-Generated O2 Binding Pocket in PqqC, a Cofactorless Oxidase Catalyzing the Final Step in PQQ Production

“…In both HPAO and GO, the reduction of O 2 is, thus, believed to occur via stepwise electron and proton transfers, as proposed in Scheme for PqqC. Work by others on flavin-containing oxidases indicates pathways for O 2 binding, as well as support for the importance of electrostatic catalysis − .…”