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Structural basis for substrate specificity in human monomeric carbonyl reductases.

Pilka ES, Niesen FH, Lee WH, El-Hawari Y, Dunford JE, Kochan G, Wsol V, Martin HJ, Maser E, Oppermann U - PLoS ONE (2009)

Bottom Line: In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids.One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum.A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Headington, United Kingdom.

ABSTRACT

Unlabelled: Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3.

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Comparison of active site properties of human CBR1 and CBR3.A–C: CBR1 (green), D–E: CBR3, with modelled loop (orange). First Column (A and D): solvent accessible surface representation of the active site pockets coloured according to electrostatic potentials, with the cofactor represented as sticks. Yellow line marks the plane cutting through the active site. The plane divides the pocket into two halves that are depicted in the following two columns. Second column (B and E): ‘left’ half of the pocket. Third column (C and F): ‘right’ half of the pocket. Cofactor is shown for orientation purpose. Residues that were mutated in this study are marked with asterisks. Catalytic residue labels are underlined.
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pone-0007113-g004: Comparison of active site properties of human CBR1 and CBR3.A–C: CBR1 (green), D–E: CBR3, with modelled loop (orange). First Column (A and D): solvent accessible surface representation of the active site pockets coloured according to electrostatic potentials, with the cofactor represented as sticks. Yellow line marks the plane cutting through the active site. The plane divides the pocket into two halves that are depicted in the following two columns. Second column (B and E): ‘left’ half of the pocket. Third column (C and F): ‘right’ half of the pocket. Cofactor is shown for orientation purpose. Residues that were mutated in this study are marked with asterisks. Catalytic residue labels are underlined.

Mentions: The main distinguishing feature of the crystal structures of human/porcine CBR1 (ternary cofactor inhibitor complex PDB 1wma [12]; binary cofactor complex PDB 1n5d [20]) and human CBR3 (binary cofactor complex, PDB 2hrb) is the conformation of the substrate binding loop: whereas the CBR1 structures show a conformation with a more closed active site, in CBR3 the loop is engaged in crystal contacts with a symmetry related molecule (Figures 2A and 2B). Despite extensive crystal screening and attempts to obtain ternary complexes, we were unsuccessful in finding different crystal forms. Inspection of the “open” structure reveals that substrate docking in this conformation is not useful to produce models explaining the observed substrate features. We therefore decided to model the CBR3 sequence using the CBR1 structure as template (Figure 3), assuming a similar loop arrangement. The loop modelling results in a conformation with all residues in acceptable regions of a Ramachandran plot, moreover docking analysis with different substrates allowed us to successfully identify critical residues for substrate recognition and catalysis. A comparison of the two CBR structures in the loop-closed conformation shows a wide opening to a gorge-like active site. In the CBR1-inhibitor complex structure (1wma), the inhibitor molecule occupies large parts of the entrance and is also covered by a PEG molecule derived from crystallization. CBR1 has a slightly narrower substrate binding cleft (Figure 4) than CBR3, mainly as a result of the terminal sulf-methyl group of Met141. This residue is replaced in CBR3 by Gln142 (Figure 4F), which has a similar but not identical conformation, as observed in structures 1wma and 2hrb.


Structural basis for substrate specificity in human monomeric carbonyl reductases.

Pilka ES, Niesen FH, Lee WH, El-Hawari Y, Dunford JE, Kochan G, Wsol V, Martin HJ, Maser E, Oppermann U - PLoS ONE (2009)

Comparison of active site properties of human CBR1 and CBR3.A–C: CBR1 (green), D–E: CBR3, with modelled loop (orange). First Column (A and D): solvent accessible surface representation of the active site pockets coloured according to electrostatic potentials, with the cofactor represented as sticks. Yellow line marks the plane cutting through the active site. The plane divides the pocket into two halves that are depicted in the following two columns. Second column (B and E): ‘left’ half of the pocket. Third column (C and F): ‘right’ half of the pocket. Cofactor is shown for orientation purpose. Residues that were mutated in this study are marked with asterisks. Catalytic residue labels are underlined.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2741203&req=5

pone-0007113-g004: Comparison of active site properties of human CBR1 and CBR3.A–C: CBR1 (green), D–E: CBR3, with modelled loop (orange). First Column (A and D): solvent accessible surface representation of the active site pockets coloured according to electrostatic potentials, with the cofactor represented as sticks. Yellow line marks the plane cutting through the active site. The plane divides the pocket into two halves that are depicted in the following two columns. Second column (B and E): ‘left’ half of the pocket. Third column (C and F): ‘right’ half of the pocket. Cofactor is shown for orientation purpose. Residues that were mutated in this study are marked with asterisks. Catalytic residue labels are underlined.
Mentions: The main distinguishing feature of the crystal structures of human/porcine CBR1 (ternary cofactor inhibitor complex PDB 1wma [12]; binary cofactor complex PDB 1n5d [20]) and human CBR3 (binary cofactor complex, PDB 2hrb) is the conformation of the substrate binding loop: whereas the CBR1 structures show a conformation with a more closed active site, in CBR3 the loop is engaged in crystal contacts with a symmetry related molecule (Figures 2A and 2B). Despite extensive crystal screening and attempts to obtain ternary complexes, we were unsuccessful in finding different crystal forms. Inspection of the “open” structure reveals that substrate docking in this conformation is not useful to produce models explaining the observed substrate features. We therefore decided to model the CBR3 sequence using the CBR1 structure as template (Figure 3), assuming a similar loop arrangement. The loop modelling results in a conformation with all residues in acceptable regions of a Ramachandran plot, moreover docking analysis with different substrates allowed us to successfully identify critical residues for substrate recognition and catalysis. A comparison of the two CBR structures in the loop-closed conformation shows a wide opening to a gorge-like active site. In the CBR1-inhibitor complex structure (1wma), the inhibitor molecule occupies large parts of the entrance and is also covered by a PEG molecule derived from crystallization. CBR1 has a slightly narrower substrate binding cleft (Figure 4) than CBR3, mainly as a result of the terminal sulf-methyl group of Met141. This residue is replaced in CBR3 by Gln142 (Figure 4F), which has a similar but not identical conformation, as observed in structures 1wma and 2hrb.

Bottom Line: In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids.One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum.A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Headington, United Kingdom.

ABSTRACT

Unlabelled: Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3.

Enhanced version: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Show MeSH
Related in: MedlinePlus