Substrate Specificity for the Epoxidation of Terpenoids and Active Site Topology of House Fly Cytochrome P450 6A1

Background: There is a large inter-individual variation in cytochrome P450 2C19 (CYP2C19) activity. The variability can be caused by the genetic polymorphism of CYP2C19 gene. This study aimed to investigate the molecular and kinetics basis for activity changes in three alleles including CYP2C19*23, CYP2C19*24 and CYP2C19*25found in the Chinese population. Methods: The three variants expressed by bacteria were investigated using substrate (omeprazole and 3- cyano-7-ethoxycoumarin[CEC]) and inhibitor (ketoconazole, fluoxetine, sertraline and loratadine) probes in enzyme assays along with molecular docking. Results: All alleles exhibited very low enzyme activity and affinity towards omeprazole and CEC (6.1% or less in intrinsic clearance). The inhibition studies with the four inhibitors, however, suggested that mutations in different variants have a tendency to cause enhanced binding (reduced IC50 values). The enhanced binding could partially be explained by the lower polar solvent accessible surface area of the inhibitors relative to the substrates. Molecular docking indicated that G91R, R335Q and F448L, the unique mutations in the alleles, have caused slight alteration in the substrate access channel morphology and a more compact active site cavity hence affecting ligand access and binding. It is likely that these structural alterations in CYP2C19 proteins have caused ligand-specific alteration in catalytic and inhibitory specificities as observed in the in vitro assays. Conclusion: This study indicates that CYP2C19 variant selectivity for ligands was not solely governed by mutation-induced modifications in the active site architecture, but the intrinsic properties of the probe compounds also played a vital role.

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A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity

Archives of Biochemistry and Biophysics ◽

10.1016/s0003-9861(04)00230-9 ◽

2004 ◽

Author(s):

J RAMAN

Keyword(s):

Substrate Specificity ◽

Active Site ◽

Site Mutation ◽

Hypoxanthine Guanine Phosphoribosyltransferase

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Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues

Journal of Biological Chemistry ◽

10.1074/jbc.274.4.2344 ◽

1999 ◽

Vol 274 (4) ◽

pp. 2344-2349 ◽

Cited By ~ 125

Author(s):

Shinya Oue ◽

Akihiro Okamoto ◽

Takato Yano ◽

Hiroyuki Kagamiyama

Keyword(s):

Substrate Specificity ◽

Active Site ◽

Cumulative Effects ◽

Active Site Residues

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Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2006.08.024 ◽

2006 ◽

Vol 455 (1) ◽

pp. 61-67 ◽

Cited By ~ 16

Author(s):

Cynthia E. Hernandez ◽

Santosh Kumar ◽

Hong Liu ◽

James R. Halpert

Keyword(s):

Cytochrome P450 ◽

Crystal Structures ◽

Active Site ◽

Active Site Residues ◽

Substrate Metabolism ◽

Cytochrome P450 2B4

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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Specificity and mechanism of carbohydrate demethylation by cytochrome P450 monooxygenases

Biochemical Journal ◽

10.1042/bcj20180762 ◽

2018 ◽

Vol 475 (23) ◽

pp. 3875-3886 ◽

Cited By ~ 3

Author(s):

Craig S. Robb ◽

Lukas Reisky ◽

Uwe T. Bornscheuer ◽

Jan-Hendrik Hehemann

Keyword(s):

Cytochrome P450 ◽

Active Site ◽

Substrate Recognition ◽

High Energy ◽

Cytochrome P450 Monooxygenases ◽

P450 Monooxygenases ◽

High Energy Barrier ◽

Molecular Determinants ◽

Cytochrome P450 Family ◽

Carbohydrate Ligand

Degradation of carbohydrates by bacteria represents a key step in energy metabolism that can be inhibited by methylated sugars. Removal of methyl groups, which is critical for further processing, poses a biocatalytic challenge because enzymes need to overcome a high energy barrier. Our structural and computational analysis revealed how a member of the cytochrome P450 family evolved to oxidize a carbohydrate ligand. Using structural biology, we ascertained the molecular determinants of substrate specificity and revealed a highly specialized active site complementary to the substrate chemistry. Invariance of the residues involved in substrate recognition across the subfamily suggests that they are critical for enzyme function and when mutated, the enzyme lost substrate recognition. The structure of a carbohydrate-active P450 adds mechanistic insight into monooxygenase action on a methylated monosaccharide and reveals the broad conservation of the active site machinery across the subfamily.

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