Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF

Abstract

The terminal step of 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis is performed by NspF, a mono-oxygenase that converts o -aminophenols to the corresponding nitroso product (hydroxyanilinase activity). Previous biochemical characterization of the resting form of NspF suggested that this enzyme belonged to the coupled binuclear copper enzyme (CBC) family. Another member of this enzyme family, tyrosinase, is able to mono-oxygenate monophenols (monophenolase activity) but not o -aminophenols. To gain insight into the unique reactivity of NspF, we have generated and characterized the oxy form of its active site. The observation of spectral features identical to those of oxy-tyrosinase indicates that oxy-NspF contains a Cu 2 O 2 core where peroxide is coordinated in a μ - η 2 ∶ η 2 mode, confirming that NspF is a CBC enzyme. This oxy form is found to react with monophenols, indicating that, like tyrosinase, NspF also possesses monophenolase activity. A comparison of the two electrophilic mechanisms for the monophenolase and hydroxyanilinase activity indicates a large geometric change between their respective transition states. The potential for specific interactions between the protein pocket and the substrate in each transition state is discussed within the context of the differential reactivity of this family of enzymes with equivalent μ - η 2 ∶ η 2 peroxy bridged coupled binuclear copper active sites.

Copper Active Sites in Biology

“…Comparison of the hydroxyanilinase versus CaOx activities in NspF versus a close homolog GriF and Ty revealed that the difference in selectivity leading to the oxygenated nitrosophenol product was a result of differences in the oxygenase (hydroxyanilinase) activity rather than differences in the oxidase (CaOx) activity (see Section 3.2.2). 497 This observation was suggestive of protein secondary coordination impacts on the reaction landscape rather than intrinsic differences in the reactivity of the Cu 2 O 2 cores, which appear indistinguishable spectroscopically. The reaction trajectories were evaluated by DFT and further supported this secondary coordination environment control hypothesis, in that the structural requirements for the hydroxyanilinase reaction differed from that of either oxygenation of monophenols or oxidation of diphenols (Figure 77).…”

“…From the magnitude of the rates, the ΔΔG ‡ for this activity in NspF relaive to Ty is expected to be greater than 4 kcal mol −1 . 497 …”

Spectroscopic and computational insight into the activation of O 2 by the mononuclear Cu center in polysaccharide monooxygenases

“…Several classes with either binuclear or trinuclear copper active sites have been identified and their different strategies for O 2 activation have been elucidated (1). These include the multicopper oxidases that use three Cu ions to reduce O 2 to water with very little overpotential (2,3), the coupled binuclear Cu enzymes that are involved in dioxygen transport and monooxygenase reactivity (4), and the noncoupled binuclear Cu monooxygenases that activate O 2 for hydroxylation of peptides and hormones (5). Recently, a class of oxygen activating enzymes with a single copper center has been identified, the polysaccharride monooxygenases [PMOs; often termed lytic polysaccharide monooxygenases (LPMOs), reflecting their ability to break polysaccharides chains and loosen crystalline structure] (6-8), or AA9 to 11 enzymes (AA = auxiliary activity) in the Carbohydrate-Active enZYmes (CAZy) database (9).…”

A Bioinspired Catalytic Oxygenase Cascade to Generate Complex Oxindoles

“…Interestingly, another oxygenase enzyme, NspF, was recently reported to follow similar final steps in oxygenation of o-aminophenol to nitrosophenol. [23] Aldol addition of the enolate of ketoester 15 to the newly generated aldehyde forms the intermediate 21. A sixmembered transition state, stabilized by hydrogen-bonding between the newly formed hydroxy group and the enolate, avoids the steric hinderance between two ester moeities.…”

“…Dimethyl-Box (E) did not provide any oxidation reaction (entry 21), thus indicating the significance of pyridine ligation in the copper complex. CH 2 Cl 2 was the best solvent among those tested (entries [22][23][24]. For its commercial availability, we preferred enantiopure A over the equally effective (rac)-A for synthesis of 13 and further optimization of the oxygenase cascade.…”