Multifaceted personality and roles of heme enzymes in industrial biotechnology

Abstract

Heme enzymes are the most prominent category of iron-containing metalloenzymes with the capability of catalyzing an astonishingly wide range of reactions like epoxidation, hydroxylation, demethylation, desaturation, reduction, sulfoxidation, and decarboxylation. Various enzymes in this category are P450s, heme peroxidases, catalases, myoglobin, cytochrome C, and others. Besides this, the natural promiscuity and amenability of these enzymes to protein engineering and evolution have also added several non-native reactions such as C-H, N-H, S-H insertions, cyclopropanation, and other industrially important reactions to their capabilities. Surprisingly, all of these reactions and their wide substrate scopes are attributed to changes in the active site scaffold of different heme enzymes as the center of all enzymes is constituted by a porphyrin ring containing iron. Multiple prominent research groups across the world, including 2018, Nobel Laureate Frances Arnold's group, have shown keen interest in engineering and evolving these enzymes for utilizing their industrial potential. Besides engineering the active site, researchers have also explored the possibility of these enzymes catalyzing non-native reactions by replacing the center porphyrin ring with other cofactors or by changing the iron in the porphyrin ring with other metal ions along with engineering the active site and thereby creating novel artificial metalloenzymes. Thus, in this mini-review from our group, for the first time, we are trying to catalog various activities catalyzed by heme enzymes and their engineered variants and their active usage in various industries along with shedding light on their potential for use in various applications in the future.

Keywords: Artificial metalloenzymes; Biocatalyst; Catalase; Green chemistry; Heme; Industrial biotechnology; Myoglobin; P450; Peroxidase.

Fig. 1

Structure of heme (iron–protoporphyrin IX). There are multiple different types of heme porphyrin rings; however, most of the metalloenzymes contain heme b. The central iron is coordinated to four nitrogen atoms of the pyrrole ring and one active site residue as an axial ligand. The 6th site of the iron is free and coordinates with incoming substrates and catalyzes the chemical reactions. The scaffold of the pyrrole ring plays a role in fitting the porphyrin ring in the active site of the enzymes and interacting with the amino acids of the active site to create an appropriate environment and thermodynamics for the reactions to proceed

Fig. 2

Catalytic cycle of various heme-containing enzymes. Different heme enzymes although contain heme porphyrin ring with central iron as a catalyst but their catalytic reactions and substrate scopes vary largely. This variability is attributed to the phenomenon of heme ligation control of the P450 FeIII/FeII reduction potential. The activity and substrate scope of these enzymes are defined by the reduction potential of the central iron and the size and spatial orientation of the active site residues of the enzyme. The reduction potential of the central iron also varies from enzyme to enzyme because of difference in interacting active site residues and mainly because of change in axial ligands, which is active site amino acid residue coordinating with the central iron of the heme ring at the 5th position leaving the 6th position vacant for interacting with incoming substrates

Fig. 3

Heme enzymes in various industries. Heme enzymes are the most prominently used enzymes in various industries because of their capability to catalyze a wide variety of reactions and their amenability to evolve using protein engineering and thereby being able to catalyze non-native reactions with wide substrate scopes. Given are a few examples of their use in various industries

Fig. 4

a–d Examples of the use of microbial P450s in the synthesis of various active pharmaceutical ingredients. a Hydroxylation of cortexolone to hydrocortisone by marine fungus Curvularia sp. b Streptomyces sp. is used for late-stage functionalization of pravastatin from compactin. c Synthetic precursor production of perillyl alcohol from limonene using CYP153 family of Pseudomonas sp. d The CYP107A1 (EryF) and CYP113A1 (EryK) from Saccharopolyspora erythraea are used for the production of the antibacterial agent erythromycin

Fig. 5

Heme peroxidase-catalyzed phenol oxidation. One important reaction catalyzed by heme peroxidases is the phenol oxidation and polymerization reaction. The reaction proceeds through the generation of ferryl intermediates (Fe(IV)=O), as potent oxidants (Compound I) followed by oxidation of phenol and radical coupling and polymerization. (Torres-Duarte and Vazquez-Duhalt 2010)

Fig. 6

Engineered myoglobin-based industrially important biotransformation reactions. Although the native activity of myoglobin is carrying and storage of oxygen, engineered and artificial myoglobin have shown immense potential in carrying out various important industrial reactions with wide substrate scope. The product formed is a high-value product with significant yields and enantioselectivity which makes myoglobin an extremely attractive biocatalyst for the industry