stucklike glu:glutamyltrna...
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Stuck Like Glu: Glutamyl-‐tRNA Reductase (GluTR) East Brunswick High School
Abstract Glutamyl-‐tRNA reductase (GluTR) catalyzes the reducBon of glutamyl-‐tRNAGlu into glutamate-‐1-‐semialdehyde (GSA). We explored the structure and funcBon of Arabidopsis thaliana GluTR and its sBmulator protein, GluTR binding protein (GluBP), to infer the role of GluTR in the aquaBc duckweed plant, Landol0a punctata.
IntroducBon Tetrapyrroles are a class of chemical compounds useful in many biological processes. Examples of tetrapyrroles include chlorophyll, heme, and phytochromobilin. 5-‐Aminolevulinic acid (ALA) is the universal precursor for tetrapyrrole biosynthesis. In plants, algae, and most bacteria, ALA is produced in a 2-‐step mechanism (see Figure 1). Stroma-‐localized GluTR catalyzes the first, rate-‐limiBng step of that mechanism.
Informally, GluTR acBvity divides into two steps:(1) glutamate is extracted from the glutamyl-‐tRNAGlu and converted into a thioester intermediate and (2) the thioester intermediate is reduced into GSA. Thylakoid membrane-‐bound GluBP sBmulates GluTR catalyBc acBvity by causing a conformaBonal change that favors electron transfer.
Methods Using BLAST analysis, students inferred the funcBon of over 100 cDNA sequences from L. punctata. We selected a sequence (clone 04SH1.14) coding for part of a domain on GluTR to further invesBgate. AXer using similar sequences to infer the protein sequence that the transcript encoded, we researched the protein’s structure, funcBon, and applicaBons.
Discussion GluTR is a homodimer, in which each subunit contains 3 domains: an N-‐terminal catalyBc domain where glutamyl-‐tRNAGlu and GluBP bind, an NADPH-‐binding domain, and a C-‐terminal dimerizaBon domain that holds the dimer together.
Figure 1: 5-‐Aminolevulinic Acid (ALA) is produced in a 2-‐step mechanism. First, glutamyl-‐tRNAGlu is reduced into GSA by GluTR in a reacBon which GluBP sBmulates. Then, GSAM isomerizes GSA into ALA, which can be converted into tetrapyrroles.
Figure 2: GluTR also funcBons in 2 steps. In the first step, it extracts glutamate from glutamyl-‐tRNAGlu, creaBng a thioester intermediate whose thioester group is labeled in red. Then, the thioester intermediate is reduced into GSA.
Figure 3: GluTR in complex with its sBmulator protein, GluBP. Both proteins are homodimers. GluTR monomers each have 3 domains and GluBP monomers each have two domains.
Discussion The reacBon starts when glutamyl-‐tRNAGlu binds to the catalyBc domain on each monomer of GluTR. The negaBvely charged tRNAGlu is a]racted to a posiBve region on GluTR. A conserved arginine (Arg415) also recognizes the tRNAGlu and another conserved arginine (Arg146) recognizes the glutamate.
Figure 4: NegaBvely charged tRNAGlu (purple) superimposed on posiBvely charged region where it binds to on GluTR (blue)
Then, Cys144 separates the glutamate from the tRNAGlu by breaking the aminoacyl bond between glutamate and the tRNAGlu, forming the thioester intermediate at the GluTR catalyBc domains. Meanwhile, NADPH binds to the NADPH-‐binding domains. Next, GluBP binds to GluTR, stabilizing the V-‐structure of GluTR and driving a conformaBonal change that twists the spinal α-‐helix on each GluTR subunit. This swings the NADPH-‐binding domains toward the catalyBc domains, making it easier for NADPH to transfer electrons to the thioester intermediate.
Figure 5: Thioester intermediate (yellow sBck) receiving electrons from NADPH (other sBck model).
Once NADPH reduces the thioester intermediate into GSA, the product is then shu]led out towards the center of the complex as a result of GSA interacBons with Gly101, Cys144, Arg146, and His193 on GluTR.
AXer being channeled, GSA is held in an exit pocket on GluTR consisBng of: His105, Glu148, Phe183, and Asp202. GSA is shielded by Lys271 on GluBP to prevent it from escaping. When GluBP detaches, GSA-‐2,1-‐aminomutase (GSAM), the next enzyme in the 2-‐step ALA synthesis mechanism, binds to a similar region on GluTR. Since the channel is no longer shielded, GSAM accepts GSA and isomerizes it into ALA.
Figure 6: Review of ALA synthesis pathway
Conclusion
Figure 7: (a) Coupled enzyme assay reveals 3-‐fold increase in GluTR acBvity by GluBP binding (b) Coupled enzyme assay reveals heme is a negaBve allosteric regulator of GluTR (c) Northern blot analysis shows gene coding for GluTR is only expressed during light exposure
Many other molecules and condiBons affect GluTR acBvity. Coupled enzyme assays reveal that GluBP increases GluTR acBvity 3-‐fold. Heme negaBvely allosterically regulates GluTR, which allows cells to conserve both energy and resources by prevenBng the producBon of excess ALA. Northern blot analysis indicates that increasing light and temperature increases GluTR producBon. Therefore, GluTR is most acBve in the presence of GluBP, low heme content, high temperature, and increased light exposure.
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References h]p://www.ncbi.nlm.nih.gov/pmc/arBcles/PMC402005, h]p://www.ncbi.nlm.nih.gov/pmc/arBcles/PMC125327/, h]p://www.jbc.org/content/274/43/30679.long, h]p://www.ncbi.nlm.nih.gov/pubmed/15757895, h]p://www.ncbi.nlm.nih.gov/pubmed/22180625, h]p://www.plantcell.org/content/6/2/265.full.pdf
Step 1: Glu ExtracBon Step 2: ReducBon
L L L L D D glutamyl-‐tRNAGlu
GSA
ALA
Tetrapyrroles (chlorophyll, heme, etc.)
GSAM
GluTR GluBP
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