Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme

Abstract

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1-C2=O locus via interaction with the hydroxyl group at C2' of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.

Scheme 1

Figure 1

(A) Amino acids in the proximity of the N1–C2=O region of the isoalloxazine ring system of wild-type BBE. (B) For comparison, the same region is shown for the His174Ala variant protein structure, showing the sideways movement of the flavin ring as indicated by the strengthened hydrogen bond to Tyr456 and the Phe180 main chain carbonyl. The electron density shown in panel B represents the 2 F_o – F_c map contoured at 1.5 σ. Distances for hydrogen bonds are given in angstroms.

Figure 2

Absorption spectra of the His174Ala variant in comparison to the wild-type enzyme. Absorption spectra of the native enzymes (A) and the enzymes after heat denaturation (B). Solid lines represent data for the wild-type enzyme in its native and denatured form. Dashed lines represent data for the His174Ala variant. All spectra are normalized to a protein concentration of ∼10 μM.

Figure 3

Anaerobic photoreduction and reoxidation of BBE His174Ala. (A) Selected spectra of the complete photoreduction. Solid lines represent the spectrum prior to illumination and the spectrum of the fully reduced flavin cofactor. Dotted lines are selected spectra recorded during the course of photoreduction. The hypsochromic shift in the maximum of the fully reduced species to 350 nm is indicated by a black arrow. (B) Selected spectra recorded upon admission of oxygen: (−·−) spectrum of the fully reduced flavin cofactor after photoillumination, (···) spectrum after a 5 s exposure to oxygen, and (—) characteristic spectrum of BBE His174Ala after reoxidation. (C) Slow regeneration of the initial absorption spectrum. Solid lines are spectra of the flavin cofactor 5 min and 5 h after the admission of oxygen. Dashed lines show spectral changes that indicate a slow regeneration of the flavin cofactor.

Figure 4

Absorption spectrum of completely reduced and consecutively denatured His174Ala. The black line is the spectrum of BBE His174Ala denatured in its completely reduced form, whereas the gray line is the absorption spectrum of denatured Cys166Ala.

Figure 5

Structural changes due to His174Ala substitution observed in the proximity of the isoalloxazine ring system. Wild-type carbon atoms are colored gray, and the corresponding elements of the His174Ala structure are colored green. Because of the missing imidazole ring of His174, a flipped peptide bond is observed between Ala163 and Gly164. The corresponding amide proton then provides one ligand for a newly formed chloride binding site. Distances for the partial octahedral coordination of the anion are given in angstroms. Water molecules colored red belong to His174Ala, and the one colored violet belongs to the wild-type protein. The bound chloride ion is colored green.

Figure 6

Additional structural changes due to altered amino acid side chains involved in the formation of the chloride ion coordination shell. With Phe332 as a starting point, these changes extend via Met182 all the way to the active site amino acid Trp165, which is in direct contact with the substrate (S)-reticuline. Wild-type carbon amino acids are colored gray, and the corresponding amino acids of the His174Ala variant are colored green.

Scheme 2