Abstract
Glutamate-1-semialdehyde-2,1-aminomutase (GSAM) catalyzes the isomerization of glutamate-1-semialdehyde (GSA) to 5-aminolevulinate (ALA) and is distributed in archaea, most bacteria and plants. Although structures of GSAM from archaea and bacteria have been resolved, a GSAM structure from a higher plant is not available, preventing further structure-function analysis. Here, the structure of GSAM from Arabidopsis thaliana (AtGSA1) obtained by X-ray crystallography is reported at 1.25 Å resolution. AtGSA1 forms an asymmetric dimer and displays asymmetry in cofactor binding as well as in the gating-loop orientation, which is consistent with previously reported Synechococcus GSAM structures. While one monomer binds PMP with the gating loop fixed in the open state, the other monomer binds either PMP or PLP and the gating loop is ready to close. The data also reveal the mobility of residues Gly163, Ser164 and Gly165, which are important for reorientation of the gating loop. Furthermore, the asymmetry of the AtGSA1 structure supports the previously proposed negative cooperativity between monomers of GSAM.
Highlights
Tetrapyrroles such as chlorophyll and haem are cofactors that are essential for a wide variety of crucial biological processes, including photosynthesis and respiration (Mochizuki et al, 2010). 5-Aminolevulinic acid (ALA) is the universal precursor of tetrapyrroles (Porra, 1997; Reinbothe & Reinbothe, 1996; von Wettstein et al, 1995)
The activated glutamate is first reduced to glutamate-1-semialdehyde (GSA) by the NADPH-dependent glutamyl-tRNA reductase (GluTR; EC 1.2.1.70; Moser et al, 1999), and GSA is isomerized to ALA by glutamate-1semialdehyde-2,1-aminomutase (GSAM; EC 5.4.3.8; Ilag & Jahn, 1992)
The gene for AtGSA1 (AT5G63570) lacking the plastidtargeting sequences was amplified by PCR from cDNA using the following primers containing sequences corresponding to the Tobacco etch virus (TEV) protease recognition site and restriction sites (BamHI and XhoI; underlined): sense primer, 50-CCTGGATCCGAAAACCTGTATTTTCAGGGCGTCGACGAGAAGAAGAAAAGTT-30; antisense primer, 50-CCTTTCTCGAGCTAGATCCTACTCAGTACCCTCTCA30
Summary
Tetrapyrroles such as chlorophyll and haem are cofactors that are essential for a wide variety of crucial biological processes, including photosynthesis and respiration (Mochizuki et al, 2010). 5-Aminolevulinic acid (ALA) is the universal precursor of tetrapyrroles (Porra, 1997; Reinbothe & Reinbothe, 1996; von Wettstein et al, 1995). The activated glutamate is first reduced to glutamate-1-semialdehyde (GSA) by the NADPH-dependent glutamyl-tRNA reductase (GluTR; EC 1.2.1.70; Moser et al, 1999), and GSA is isomerized to ALA by glutamate-1semialdehyde-2,1-aminomutase (GSAM; EC 5.4.3.8; Ilag & Jahn, 1992). ALA formation is the rate-limiting step in tetrapyrrole biosynthesis (Tanaka & Tanaka, 2007). GSAM, named glutamate-1-semialdehyde aminotransferase (GSA-AT), is a pyridoxamine 50-phosphate (PMP)/pyridoxal 50-phosphate (PLP)-dependent enzyme. Almost all B6 cofactors, including PLP and PMP, depend on the pyridinium moiety to stabilize high-energy anionic intermediates during reaction (Agnihotri & Liu, 2001). GSAM catalyzes the transamination of GSA substrate to ALA product by an unusual intramolecular exchange of amino and oxo groups via the intermediate 4,5-diaminovalerate (DAVA). The reaction starts with imine formation between PMP and the aldehyde of GSA (Fig. 1, step 1). The double bond of this imine shifts to yield an
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