Dihydrofolate reductase

From Proteopedia

(Difference between revisions)
Jump to: navigation, search
Current revision (11:22, 21 January 2024) (edit) (undo)
 
(177 intermediate revisions not shown.)
Line 1: Line 1:
-
[[Image:1rx2.jpg|left|150px]]<br />
+
[[Image:34-DihydrofolateReductase-3dfr-1dls.PNG|thumb|350px|E. coli (left) and human (right) DHFR have a similar architecture and mode of binding to NADPH(green) and the competitive inhibitor methotrexate(purple). Original image by David Goodsell]]
-
<applet load='1rx2' size='300' color='white' frame='true' spin='on' caption='DHFR' align='right' />
+
The enzyme [[dihydrofolate reductase]] (DHFR) occurs in all organisms and has been particularly well-studied in the bacterium Escherichia coli and in humans<ref>DOI:10.1146/annurev.biophys.33.110502.133613</ref><ref>https://en.wikipedia.org/wiki/Dihydrofolate_reductase</ref><ref>https://pdb101.rcsb.org/motm/34</ref>. It catalyzes the reduction of dihydrofolate to tetrahydrofolate, with NADPH acting as hydride donor. The human enzyme is a target for developing inhibitors used in anti-cancer chemotherapies<ref>doi:10.3390/molecules24061140</ref>, while the bacterial enzymes are targets for developing inhibitors as antibiotics. DHFR is a model enzyme for studying the kinetics, mechanism, and inhibition of enzymatic reactions and the underlying structure and conformational dynamics.
-
'''3D structure of dihydrofolate reductase'''<br />
+
-
== The sole source of tetrahydrofolate==
+
-
DHFR is a ubiquitous enzyme found in all organisms. The primary physiological role of DHFR is maintenance of the intracellular levels of tetrahydrofolate, a precursor of cofactors required for the biosynthesis of purines, pyrimidines, and several amino acids.
+
== DHFR occurs in all organisms and most cells==
-
The enzyme, which is the sole source of tetrahydrofolate catalyzes the reduction of <scene name='Dihydrofolate_reductase/Dihydrofolate/1'>7,8-dihydrofolate</scene> (DHF) to 5,6,7,8-tetrahydrofolate (THF) by stereospecific hydride transfer from the <scene name='Dihydrofolate_reductase/Nadph/2'>NADPH</scene> cofactor to the C6 atom of the pterin ring with concomitant protonation at N5.
+
DHFR is found in all organisms. Some bacteria acquire resistance to DHFR inhibitors through expressing a second form of DHFR coded on a plasmid. The enzymes from E. coli (ecDHFR) and humans (hDHFR) have similar folds, while the plasmid-encoded enzyme has an unrelated fold. In humans, DHFR is expressed in most tissues[https://www.proteinatlas.org/ENSG00000228716-DHFR], and there are two genes, DHFR and DHFR2/DHFRL1, the latter targeted to mitochondria<ref>DOI:10.1073/pnas.1103605108</ref>. Mice and rats lack the second gene but also show DHFR activity in mitochondria<ref>DOI:10.1016/j.febslet.2015.05.017</ref>.
-
Being the sole source of THF it is an Achilles’ heel of rapidly proliferating cells, making it an attractive target of several important anticancer and antimicrobial drugs such as methotrexate, trimethoprim, and pyrimethamine.
+
-
''E. coli'' DHFR is a small (18 kD) protein with an α/β structure consisting of a central eight-stranded β-sheet and four flanking α helices. The active site cleft divides the protein into two structural subdomains: the <scene name='Dihydrofolate_reductase/Adenosine_sub/2'>Adenosine binding subdomain</scene> (red) and the <scene name='Dihydrofolate_reductase/Adenosine_sub/3'>major subdomain</scene> (green). The adenosine binding subdomain is the smaller of the two subdomains and provides the binding site for the adenosine moiety of the cofactor. The major subdomain consists of ~100 residues from the N and C termini and is dominated by a set of three loops on the ligand binding face that surround the active site. In terms of sequence length, these loops make up approximately 40%–50% of the major subdomain; hence it is sometimes called the “loop” subdomain. The loops are termed Met20, F-G, and G-H loops. Hinge bending motions about Lys38 and Val88 allow the adenosine binding domain to move relative to the major domain upon binding of various ligands, resulting in closure of the active site cleft.
+
== Reactions catalyzed ==
 +
[[Image:DHFR_reaction.gif|The reaction catalyzed by DHFR reduces a double bond in dihydrofolate (DHF) to form tetrahydrofolate (THF) by transfering a hydride from nicotinamide adenine dinucleotide phosphate (NADPH)]]
-
[[Image:Met20loop.png|left|250px]]<br />
+
The reaction catalyzed by DHFR reduces a double bond in dihydrofolate (DHF) to form tetrahydrofolate (THF) by transfering a hydride from nicotinamide adenine dinucleotide phosphate (NADPH)
-
A movie depicting the conformational changes of DHFR during one turnover of substrate has been constructed by M. R. Sawaya and J. Kraut using six isomorphous crystallographic structures.
+
[[Dihydrofolate reductase]] (DHFR, 1.5.1.3 [https://enzyme.expasy.org/EC/1.5.1.3]) is an enzyme which uses the co-factor NADPH as electron donor. It catalyzes the reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) as NADPH is oxidized to NADP+. The mammalian enzymes also accept folic acid as a substrate, reducing it to THF. This allows the use of folic acid, which is easier to synthesize than DHF or THF, to fortify food.<ref>DOI:10.3746/pnf.2014.19.4.247</ref><ref>https://ods.od.nih.gov/factsheets/Folate-HealthProfessional/</ref>. Some bacterial enzymes also accept folic acid as a substrate <ref>DOI:10.1021/acs.biochem.6b01268</ref> but it acts as a competitive inhibitor in the E. coli enzyme.
-
http://chem-faculty.ucsd.edu/kraut/dhfr.html
+
 
 +
[[Image:467px-DHFR rxn.svg.png|300px]]
 +
 
 +
The folate is a form of the essential vitamin B9. Folate is not part of our natural diet (it contains dihydrofolate and tetrahydrofolate, sometimes as a poly-glutamate conjugate) but is bioavailable and simpler to synthesize.
 +
 
 +
== Relevance ==
 +
Tetrahydrofolate (THF) is an essential cofactor of [[one-carbon metabolism]]<ref>PMID:18804690</ref><ref>doi:10.1007/978-1-4020-2400-9_12</ref>. [[Image:one-carbon.PNG|thumb|Metabolites and enzymes of one-carbon metabolism. Metabolites shown in red (DHF, 10f-THF, CH=THF, CH2-THF, 5mTHF) are all related to THF (by oxidation or methylation)]]For example, THF is required for turning homocysteine into the amino acid methionine, and for biosynthesis of dTTP, one of the four nucleotide building blocks of DNA, from dUTP. In these reactions, tetrahydrofolate is first methylated and then oxidized to dihydrofolate. To allow for multiple rounds of turnover, dihydrofolate has to be reduced again; dihydrofolate reductase is the enzyme that enables this. Like many common cofactors, tetrahydrofolate is not synthesized de novo in the human body. Instead, it is provided as vitamin B9 in a healthy diet, for instance through leafy vegetables (the name for folic acid come from e.g. Latin folio, leaf). Many countries fortify common food ingredients like flour with vitamin B9 (in the form of folic acid) to ensure sufficient dietary levels. As described above, DHFR also plays a role in reducing folic acid to the biologically active tetrahydrofolate.
 +
 
 +
== Structure and Function ==
 +
<StructureSection load='' size='350' side='right' caption='' scene='82/82636/Dhfr_ternary/4'>__NOTOC__
 +
=== Fold and ligand binding ===
 +
DHFR from bacteria and vertebrates share a common fold ([http://www.cathdb.info/version/latest/superfamily/3.40.430.10 CATH], [https://scop.mrc-lmb.cam.ac.uk/term/3001255 SCOP]) featuring a <scene name='82/82636/Fold/5'>central beta sheet</scene> surrounded by <scene name='82/82636/Fold/6'>loops and alpha helices</scene>. The <scene name='82/82636/Fold/7'>ligands</scene> bind in similar locations in bacterial and vertebrate enzymes. The substrate (and the competive inhibitors <jmol>
 +
<jmolLink>
 +
<script>
 +
define current selected;
 +
select MTX;
 +
selectionHalos on;
 +
delay 0.5;
 +
selectionHalos off;
 +
select current;
 +
</script>
 +
<text>☼</text>
 +
</jmolLink>
 +
</jmol>) bind on one side of the beta sheet while the co-factor NADPH <jmol>
 +
<jmolLink>
 +
<script>
 +
define current selected;
 +
select NDP;
 +
selectionHalos on;
 +
delay 0.5;
 +
selectionHalos off;
 +
select current;
 +
</script>
 +
<text>☼</text>
 +
</jmolLink>
 +
</jmol> wraps around the sheet (around the C-terminal ends of strands as found in the classic [[Rossmann fold]] nucleotide binding site), with the nicotinamide moiety next to the substrate and the adenine moiety on the other side of the sheet.
 +
 
 +
<jmol>
 +
<jmolRadioGroup>
 +
<item>
 +
<script>anim off; delay 1.0; model 1</script>
 +
<text>bacterial (L. casei)</text>
 +
<checked>true</checked>
 +
</item>
 +
<item>
 +
<script>anim off; delay 1.0; model 2</script>
 +
<text>human</text>
 +
<checked>false</checked>
 +
</item>
 +
<item>
 +
<script>anim fps 1; anim mode loop; anim on;</script>
 +
<text>animate</text>
 +
<checked>false</checked>
 +
</item>
 +
</jmolRadioGroup>
 +
</jmol>
 +
 
 +
In some cases, DHFR forms a complex with the enzymes that produce dihydrofolate, e.g. thymidylate synthase (TS)<ref>PMID:2178951</ref>.
 +
=== Mechanism ===
 +
 
 +
DHFR is thought to proceed in a multi-step mechanism. Once NADPH and DHF are bound to the E. coli enzyme, DHF is first protonated and then reduced through a hydride transfer from NADPH.<ref>doi:10.1073/pnas.1415940111</ref> Substrate binding and product release is thought to have a definite choreography, with fresh NADPH binding before THF is released.
 +
 
 +
 
 +
The <scene name='82/82636/Substrate_product/5'> domain orientation and the methionine 16 loop conformations</scene> changes during the catalytic cycle. The enzyme has an occluded conformation where a loop blocks entry of NADPH into the active site, and a closed conformation, where NADPH and DHF are in close contact.
 +
{{Template:Button Toggle Animation2}}
 +
 
 +
 
 +
A model by M. R. Sawaya and J. Kraut from 1997 based on six isomorphous crystal structures shows the envisioned sequence of events in the movie below <ref>doi:10.1021/bi962337c</ref>. The original web page of this movie is available on the [https://web.archive.org/web/19991003061611/http://chem-faculty.ucsd.edu/kraut/dhfr.html web archive].
 +
 
 +
[[Image:Dhfr.movie2.gif]]
 +
 
 +
The <scene name='82/82636/Active_site/1'>protonation step</scene> was studied using neutron diffraction and high-resolution X-ray diffraction to visualize the hydrogen atoms that are usually not visible in crystal structures.<ref>doi:10.1073/pnas.1415856111</ref>
 +
 
 +
The hydride transfer is thought to involve hydride tunneling, supported by temperature-dependent kinetic isotope effects<ref>doi:10.1021/ja209425w</ref>. Tunneling is a quantum phenomenon explaining how a small particle can cross an activation barrier even when it lacks sufficient activation energy. <ref>doi:10.3390/quantum3010006</ref>
 +
 
 +
=== Inhibitors ===
 +
 
 +
The human enzyme is sufficiently different from the bacterial enzymes to make specific inhibitors<ref>DOI:10.1038/s41429-019-0240-6</ref>. Inhibitors of the human enzyme are used in cancer treatment while inhibitors of the bacterials enzymes<ref>PMID:27352799</ref> are used as broad-spectrum antibiotics or more specifically to treat a specific infectious disease. An example of in inhibitor of bacterial DHFR is [[Trimethoprim]] (TMP).
 +
 
 +
Antifolate inhibitors like <scene name='82/82636/Mtx_inhibitor/2'>Methotrexate</scene> (MTX), which is very similar to <scene name='82/82636/Substrate_product/1'>dihydrofolate and tetrahydrofolate</scene>, are used in cancer therapy, see also [[Methotrexate]].
 +
 
 +
=== Resistance ===
 +
Antibiotics that inhibit bacterial DHFR are widely used in medicine and agriculture, and some bacterial strains have acquired resistance<ref>DOI:10.3109/14756366.2013.787422</ref>. For example, some bacteria carry (either on their genome or on a plasmid) the trimethoprim-resistant ''dfrB'' gene coding for an enzyme DfrB that has a fold distinct from DHFR (coded by the ''folA'' gene)<ref>PMID:7583655</ref>. Like DHFR, Dfrb has dihydrofolate reductase activity, but unlike DHFR, it is not inhibited by trimethoprim. Thus, resistant bacteria can grow even in the presence of [[Trimethoprim]], relying on the Dfrb enzyme while the DHFR enzyme is inhibited. The <scene name='82/82636/Dfrb/1'>Dfrb structure</scene> is unique in that the active site has fourfold symmetry, with DHF and NADPH bound by the same amino acid residues from different subunits. In the available crystal structure, two folate molecules are bound (use checkbox to show). The enzyme is non-specific and can bind to unproductive combinations of ligands (two DHF molecules or two NADPH molecules) or to the productive combination of one DHF and one NADPH molecule each.
 +
 
 +
<jmol>
 +
<jmolCheckbox>
 +
<scriptWhenUnChecked>hide fol or hidden
 +
</scriptWhenUnChecked>
 +
<scriptWhenchecked>select fol; wireframe 1.0; hide hidden and not fol
 +
</scriptWhenchecked>
 +
<checked>false</checked>
 +
<text>Two molecules of folate bound in the active site</text>
 +
</jmolCheckbox>
 +
</jmol>
 +
</StructureSection>
 +
 
 +
== See also ==
 +
* An overview of pathways involving folate and S-adenosyl methioine: [[One-carbon metabolism]]
 +
* Another page on DHFR: [[Molecular Playground/DHFR]]<br />
 +
* Fighting malaria with antifolates" [[Malarial Dihydrofolate Reductase as Drug Target]].<br />
 +
* Overview of enzymes targeted in cancer therapy: [[Cancer]].
 +
 
 +
 
 +
== 3D Structures of Dihydrofolate reductase ==
 +
[[Dihydrofolate reductase 3D structures]]
 +
 
 +
== Acknowledgements ==
 +
 
 +
This page was revised as part of the Spring 2022 Biochemistry II course at [https://www.westfield.ma.edu/ WSU]. Thanks go to [[User:Kia Yang|Kia]], [[User:Anna Postnikova|Anna]], [[User:Shaylie Albright|Shaylie]] and [[User:Michael O'Shaughnessy|Michael]] for helpful suggestions to improve the page.
 +
 
 +
== References ==
 +
<references/>
 +
[[Category: Dihydrofolate reductase]]
 +
[[Category:Topic Page]]
 +
[[Category:One-carbon metabolism]]

Current revision

E. coli (left) and human (right) DHFR have a similar architecture and mode of binding to NADPH(green) and the competitive inhibitor methotrexate(purple). Original image by David Goodsell
E. coli (left) and human (right) DHFR have a similar architecture and mode of binding to NADPH(green) and the competitive inhibitor methotrexate(purple). Original image by David Goodsell

The enzyme dihydrofolate reductase (DHFR) occurs in all organisms and has been particularly well-studied in the bacterium Escherichia coli and in humans[1][2][3]. It catalyzes the reduction of dihydrofolate to tetrahydrofolate, with NADPH acting as hydride donor. The human enzyme is a target for developing inhibitors used in anti-cancer chemotherapies[4], while the bacterial enzymes are targets for developing inhibitors as antibiotics. DHFR is a model enzyme for studying the kinetics, mechanism, and inhibition of enzymatic reactions and the underlying structure and conformational dynamics.

Contents

DHFR occurs in all organisms and most cells

DHFR is found in all organisms. Some bacteria acquire resistance to DHFR inhibitors through expressing a second form of DHFR coded on a plasmid. The enzymes from E. coli (ecDHFR) and humans (hDHFR) have similar folds, while the plasmid-encoded enzyme has an unrelated fold. In humans, DHFR is expressed in most tissues[1], and there are two genes, DHFR and DHFR2/DHFRL1, the latter targeted to mitochondria[5]. Mice and rats lack the second gene but also show DHFR activity in mitochondria[6].

Reactions catalyzed

The reaction catalyzed by DHFR reduces a double bond in dihydrofolate (DHF) to form tetrahydrofolate (THF) by transfering a hydride from nicotinamide adenine dinucleotide phosphate (NADPH)

The reaction catalyzed by DHFR reduces a double bond in dihydrofolate (DHF) to form tetrahydrofolate (THF) by transfering a hydride from nicotinamide adenine dinucleotide phosphate (NADPH)

Dihydrofolate reductase (DHFR, 1.5.1.3 [2]) is an enzyme which uses the co-factor NADPH as electron donor. It catalyzes the reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) as NADPH is oxidized to NADP+. The mammalian enzymes also accept folic acid as a substrate, reducing it to THF. This allows the use of folic acid, which is easier to synthesize than DHF or THF, to fortify food.[7][8]. Some bacterial enzymes also accept folic acid as a substrate [9] but it acts as a competitive inhibitor in the E. coli enzyme.

The folate is a form of the essential vitamin B9. Folate is not part of our natural diet (it contains dihydrofolate and tetrahydrofolate, sometimes as a poly-glutamate conjugate) but is bioavailable and simpler to synthesize.

Relevance

Tetrahydrofolate (THF) is an essential cofactor of one-carbon metabolism[10][11].
Metabolites and enzymes of one-carbon metabolism. Metabolites shown in red (DHF, 10f-THF, CH=THF, CH2-THF, 5mTHF) are all related to THF (by oxidation or methylation)
Metabolites and enzymes of one-carbon metabolism. Metabolites shown in red (DHF, 10f-THF, CH=THF, CH2-THF, 5mTHF) are all related to THF (by oxidation or methylation)
For example, THF is required for turning homocysteine into the amino acid methionine, and for biosynthesis of dTTP, one of the four nucleotide building blocks of DNA, from dUTP. In these reactions, tetrahydrofolate is first methylated and then oxidized to dihydrofolate. To allow for multiple rounds of turnover, dihydrofolate has to be reduced again; dihydrofolate reductase is the enzyme that enables this. Like many common cofactors, tetrahydrofolate is not synthesized de novo in the human body. Instead, it is provided as vitamin B9 in a healthy diet, for instance through leafy vegetables (the name for folic acid come from e.g. Latin folio, leaf). Many countries fortify common food ingredients like flour with vitamin B9 (in the form of folic acid) to ensure sufficient dietary levels. As described above, DHFR also plays a role in reducing folic acid to the biologically active tetrahydrofolate.

Structure and Function

Drag the structure with the mouse to rotate

See also


3D Structures of Dihydrofolate reductase

Dihydrofolate reductase 3D structures

Acknowledgements

This page was revised as part of the Spring 2022 Biochemistry II course at WSU. Thanks go to Kia, Anna, Shaylie and Michael for helpful suggestions to improve the page.

References

Retrieved from "http://52.214.119.220/wiki/index.php/Dihydrofolate_reductase"
Personal tools