Created By: Vyshnavi Pendala
NADPH-cytochrome P450 reductase or Cytochrome P411 (PDB ID: 5UCW) is an iron-containing enzymatic catalyst from the Cytochrome P450 monooxygenases family derived from
Bacillus megaterium. This biomolecule serves as an enantioselective intramolecular catalyst of benzylic C-H bonds (9). POR deficiency, specifically Cytochrome P450 oxidoreductase deficiency (PORD), is a form of congenital adrenal hyperplasia that results in a deficiency range from ambiguous and abnormal genitalia to skeletal malformations (8). The role of these reductases is being further analyzed in relation to the human body, but, synthetically, these enzymes serve as catalysts for the amination of C-H bonds. Amination of selective C-H bonds converts many chemically inert bonds in organic molecules to functionally active bonds such as C-X, C-O, C-N, or C-C bonds (9). This conversion helps expedite the synthesis of nanotechnology such as complex molecular scaffolds (1).
However, two major problems limit the amination of these ubiquitous bonds: innate selectivity and reagent/catalyst control (1). While few reagents result in a degree of regioselectivity, the quest for an enantio- and diastereoselectivity reagent remains pertinent to the reactivity of the bond (1). Many reagents tend to be precious transition metal complexes such as ruthenium, iridium, and rhodium, which are expensive and not environmentally friendly (9). Although other methods of functionalization such as the utilization of intramolecular or directing groups exist, none of these methods result in an efficient turnover along with high enantioselectivity across various substrates (9). Cytochrome P450 monooxygenases hydroxylate only 1 C-H bond in many metabolites, resulting in excellent selectivity and turnover rates. Variants in this cytochrome family such as Cytochrome P411 serve as proficient catalysts for nitrene transfer for an amination of intramolecular C-H bonds with high turnover rates and excellent selectivity (9).
Cytochrome P411 from Bacillus megaterium was expressed in Escherichia coli (E.Coli) BL21 using bacterial transformation techniques (9). The protein was crystallized through vapor diffusion sitting drop under the conditions of 0.1 M Bis-tris methane pH 5.0, 13% PEG 3350, 0.2 M NaHCOO, 22 mg/ml protein at 298.0K (10). The protein's structural data was obtained through X-Ray Crystallography (10). Ligands were not used to enhance the crystallization in this biomolecule (10).
The Expasy database, Expert Protein Analysis System containing intrinsic properties of each protein, indicated that Cytochrome P411 has a molecular weight of 107861.52 Da and an isoelectric point of 6.00 (11). Consisting of
two subunits, Cytochrome P411 is an oxidative monomeric hemoprotein consisting of 17 mutations created to enhance the amination of C-H (8, 9,11). Each subunit plays a unique role in C-H amination.
Subunit A focuses on enhancing the substrate binding ability through its conformation (8, 13).
Subunit B plays a role in the selectivity for the amination of C-H bonds (8, 13). Out of the 472 residues, residues 1-460 were crystallized in full length at 1.7Å (10). Residues 460-1100, including the C terminus, were not entirely crystallized (11). Four beneficial mutations were identified through this crystallography: B'Helix (A82L, A78V) and I Helix (F263L, E267D) (9). These residues mediated substrate binding, impacting the selectivity of the reductase (9). Four important domains are found in this protein that help reduce the iron containing heme group: NADPH binding domain, FAD binding domain, connecting domain, and FMN binding domain (6). The subunit for each corresponding domain could not be identified. NADPH binding domain binds to NADPH and oxidizes the molecule (6). The connecting domain connects the FAD and FMN binding domains together (6). The FAD binding domain binds to cofactor FAD, which serves as an electron transport protein (6). The FMN binding domain binds to cofactor FMN, which aids in the electron transfer to the heme oxygenase (6). Heme oxygenase is a catalytic enzyme that degrades the heme group into a ferrous state, thus making Cytochrome P411 active (9). A unique ligand, or cofactor, found in this protein is
Protoporphyrin IX Containing FE (HEME group) (9,10). This cofactor catalyzes the nitrene transfer responsible for the intramolecular C-H aminations (9,10).
The primary structure of Cytochrome P411 contains 944 residues in two subunits with both hydrophilic and hydrophobic amino acids (9). The
secondary structure is approximately 10%
β-sheet (12 strands, 51 residues) and 51%
α-helix (23 helices, 245 residues) in each subunit (10). The remainder of this protein was composed of random coils. The
α-helix dominated structure creates a hydrophobic core to wrap around the heme group and stabilize the structure through ionic interactions. The solvent-exposed heme group results in a formally charged ferric iron porphyrin core; however, surrounding the heme group at the center of the protein with the
α-helices stabilizes this group into a neutral ferrous heme (12). The
α-helix dominated structure suits a
mixed polarity of the sequence. Thus, hydrophobic residues primarily reside in the protein's interior or core while the exterior contains polar residues (12). The protein's structural stability is achieved through the hydrogen bonding between the carbonyl oxygen of the amino acid and the amide hydrogens in both the
α-helix and
β-sheet. Minimization of steric interference between the side chains further stabilizes the α-helix.Within the tertiary structure of Cytochrome P411, the four domains surrounding the heme group create a substrate-binding pocket (13). A majority of the encompassment is created using α-helices with few strands of β-sheets. Within the quaternary structure, the two subunits stabilize the heme group in the middle through ionic interactions between their respective α-helices and β-sheets (13). Researchers have suggested that the potential subunits create an "infinity" shape (10).
The reduction pathway begins with a molecule of NADPH binding to the NADPH binding domain found near the N terminus of the protein (5,6,9). Cofactors FMN and FAD bind to their respective domains (FAD binding domain and FMN binding domain) to serve as an intermediate for the transport of electrons from the NAPDH molecule to the heme group located at the core of the protein (5,9). These cofactor binding domains are connected to each other through the connecting domain - allowing the electron to transfer through (5,9). Once the ferric state of the heme cofactor (containing Fe
3+ ) is reduced to a ferrous state (containing Fe
2+ ), the protein reacts with a nitrene source (TsN
3 ) to create a putative iron nitrenoid (5,9). This iron nitrenoid inserts itself in a benzylic C-H bond, resulting in a benzylic amide product (5,9). If an alkane is not present for the iron nitrenoid to react with, the protein is oxidized back to its original state, and TsNH
2 is released (5,9). Amino acids
Trp-90, Thr-91, Glu-93, Thr-142, Tyr-143,
Leu-181, Glu-183, and Met-185 help bind to cofactors FMN and FAD, aiding C-H animation (7,8).
An alternate conformation of Cytochrome P411,
NADPH-P450 reductase (PDB ID:4WG2), structurally differs Cytochrome P411 due to its extra ligand,
sulfate ion along with an
additional subunit (6). The functionality of the molecule remains similar to Cytochrome P411; however, this protein aminates the C-H bond closest to its iron nitrenoid (6). Data from crystallography indicated that the secondary and tertiary structure of NADPH-P450 reductase showed
little to no difference when compared to Cytochrome P411 (6,10).
Psi-BLAST, specifically the Position-Specific-Iterated Basic Local Alignment Search Tool, database is a software that compares similar primary structures to a protein query (2). Proteins with similar primary structure as referred to as subjects (2). This software analyzes the gaps between the total sequence homology and assigns an E value per sequence (2). An E value of less than 0.05 is significant for protein analysis because a smaller E value indicates more homology while a larger E value represents more gaps within the compared structures (2). The Dali server compares the tertiary structure of proteins and assigns a respective Z-score by calculating the differences in intramolecular distances through a sum-of-pairs method (3). Structures with a Z-score greater than 2 are significant and shown to be similar (3).
Cytochrome P450 2A6 (PDB ID: 3EBS), derived from
Homo sapiens, proves to be structurally similar to Cytochrome P411 with an E value of 6 * 10
-122 along with a Z-score of 35.9 (2,3).
Both proteins serve as oxidative hemoproteins expressed in
E.coli and contain Protoporphyrin IX Containing FE ligand that becomes oxidized; however, the CYP2A contains an additional ligand:
N-(4-ethoxyphenyl) acetamide (Phenacetin) (4,10). CYP2A was synthesized with this additional ligand and its respective binding domain to compare the functionality of two proteins within Cytochrome P450 2A family (4,10). Cytochrome P450 2A6 contains
two additional subunits (4,10). Comparing CYP2A and Cytochrome P411, data indicates that similar amounts of
β-sheets and
α-helices are present within each structure. CYP2A contains 52%
α-helices (20 helices; 249 residues) and 10%
β-sheets (16 strands; 48 residues) (4,10). Although both molecules are similar in structure, their functionality varies (4,9). Cytochrome P411 is utilized synthetically, but CYP2A is found in the human body and is responsible for the metabolism of various substances such as nicotine and cocaine (4,9). Results indicated that amino acid
residues 208 300, 301, and
369 are responsible for the metabolism of Phenacetin (4,9). In totality, both proteins function similarly in the sense that reduction entails state of activity, necessary to carry out respective processes (4,9).
In conclusion, the biological significance of Cytochrome P411 lies in its ability to aminate C-H bonds. The oxidation of Cytochrome P411 through its electron donor, NADPH, activates the protein and results in the amination benzylic C-H bonds (9). Other cost-effective and environmentally friendly enzymes created via mutations are currently being tested to measure their ability to aminate C-H bonds (9). Additional research about how POR deficiency and mutations results in abnormalities in bone growth and development along with the role of Cytochrome P450 in the metabolism of drugs within the human body (4,13). Overall, the reduction pathway of Cytochrome P411 illustrates that mutated forms of this class of proteins may result in various undiscovered C-H functionalization reactions along with a deeper understanding of how protein deficiency affects the human body (4,13).