Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2.
In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in (1)H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg(246) binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-beta-lactamase superfamily.
Limphong
Pattraranee
P
Department of Chemistry and Biochemistry, 160 Hughes Hall, 701 E. High Street, Miami University, Oxford, OH 45056, USA.
Crowder
Michael W
MW
Bennett
Brian
B
Makaroff
Christopher A
CA
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000099301:Hydroxyacylglutathione Hydrolase
D029681:Arabidopsis Proteins; D007791:Lactoylglutathione Lyase; D008670:Metals; D013869:Thiolester Hydrolases; D001618:beta-Lactamases; D000099301:Hydroxyacylglutathione Hydrolase
Acta Crystallogr D Biol Crystallogr. 2006 Sep;62(Pt 9):964-70
16929096
Nat Struct Biol. 2000 Nov;7(11):1041-5
11062560
J Biol Chem. 2004 Jul 2;279(27):28653-61
15117945
Drug Resist Updat. 2000 Oct;3(5):263-264
11498393
FEBS Lett. 1997 Dec 1;418(3):351-4
9428743
J Med Chem. 1999 Jan 28;42(2):221-8
9925727
Biochem Mol Biol Int. 1995 Apr;35(4):763-71
7627127
Gen Pharmacol. 1996 Jun;27(4):565-73
8853285
J Med Chem. 1994 Jul 8;37(14):2161-6
8035422
J Biol Chem. 2005 Dec 9;280(49):40668-75
16227621
Acc Chem Res. 2006 Oct;39(10):721-8
17042472
Biochemistry. 2006 May 16;45(19):6044-55
16681377
J Biol Inorg Chem. 2004 Jun;9(4):429-38
15067523
J Biol Chem. 2002 Aug 9;277(32):29078-85
12029081
Cancer J. 2006 May-Jun;12(3):222-8
16803681
J Biol Chem. 1997 Sep 5;272(36):22502-8
9278402
Biochemistry. 1992 Oct 13;31(40):9603-8
1327121
J Clin Endocrinol Metab. 1996 Feb;81(2):488-92
8636255
Leuk Res. 1996 Jan;20(1):17-26
8632674
Biochem Soc Trans. 1993 May;21(2):545-9
8359529
J Biol Inorg Chem. 2008 Jan;13(1):15-24
17932693
J Inorg Biochem. 2008 Sep;102(9):1825-30
18656261
Trends Biochem Sci. 1998 Oct;23(10):381-2
9810225
Proc Natl Acad Sci U S A. 2004 May 18;101(20):7687-92
15128939
Biochem Soc Trans. 1993 May;21(2):531-4
8359526
Arch Biochem Biophys. 2001 Mar 1;387(1):1-10
11368170
Methods Enzymol. 1989;177:246-63
2558273
Plant Mol Biol. 1997 Nov;35(4):471-81
9349270
Am J Hum Genet. 2004 Feb;74(2):239-52
14732903
Biochem Biophys Res Commun. 1993 Apr 30;192(2):754-9
8484781
J Med Chem. 2000 Oct 19;43(21):3981-6
11052803
Cancer Chemother Pharmacol. 2000;46(2):156-66
10972486
Amino Acids. 1994 Feb;6(1):15-23
24190739
Crit Rev Oncol Hematol. 1995 Aug;20(1-2):99-128
7576201
Nature. 2007 Aug 16;448(7155):775-9
17603473
Structure. 1999 Sep 15;7(9):1067-78
10508780
Neurobiol Aging. 2007 Jan;28(1):29-41
16427160
Bioorg Med Chem Lett. 1999 Mar 22;9(6):853-6
10206549
J Biol Chem. 2001 Feb 16;276(7):4788-95
11085979
Biochem Pharmacol. 1994 Jan 20;47(2):418-20
8304987
Biochem J. 2006 Jan 1;393(Pt 1):227-34
16159313
J Clin Invest. 1998 Mar 1;101(5):1142-7
9486985
J Biol Chem. 2006 Sep 8;281(36):26702-13
16831876
Mol Aspects Med. 1993;14(4):287-371
8277832
Biochem Pharmacol. 2007 Dec 15;74(12):1686-701
17597585
Biochemistry. 2003 Oct 14;42(40):11777-86
14529289
Breast Cancer Res Treat. 2001 Mar;66(1):67-72
11368412
EMBO J. 1997 Jun 16;16(12):3386-95
9218781