Design and characterization of flavoenzyme models in the course of chemical evolution of four-α-helix bundle polypeptides

Abstract

The chemical evolution of four-α-helix bundle polypeptides to a flavoenzyme model was attempted by designing single-chain 53-peptides, which comprise flavin derivatives at three different positions. A pair of flavin derivatives were also introduced opposite to each other on the separate α-helix segments in the hydrophobic core. The four-α-helix bundle structure and the flavin moieties around the hydrophobic core were characterized by circular dichroism and fluorescence measurements, respectively. The flavoenzyme models were then examined for catalytic oxidation reaction of various N-alkyl-1,4-dihydronicotinamides (alkyl-NAHs) in aqueous solution. Since the hydrophobic core seemed to be too tightly aggregated for a catalytic group, a series of alkanesulfonates was tested to enlarge it by forming mixed micelles. The expanded hydrophobic core may more easily accommodate a hydrophobic substrate. Alkyl chains longer than dodecyl enhanced the oxidation of benzyl-NAH by a flavoenzyme model by about 6-fold. A series of alkyl-NAHs (n-butyl-, n-hexyl-, n-octyl-, n-decyl-, and n-dodecyl-NAHs) were also used as substrates in the oxidative reaction by flavoenzyme models to differentiate the positions of the flavin derivative with respect to the hydrophobic core according to their catalytic activities. The oxidation rate constants (kcat/KM) showed significant alkyl-chain-length dependence. The flavoenzyme models accommodated n-decyl- and n-dodecyl-NAHs favorably with KM values in the (1.5–5.0) × 10−5 mol dm−3 range. These results suggest that the hydrophobic core in the bundle structure is useful to position the catalytic groups and has the advantage of accommodating hydrophobic substrates. Thus, there is the possibility to utilize the hydrophobic core of the polypeptide structure to evolve the artificial proteins chemically by de novo design towards artificial enzymes.