Crystallization and preliminary X-ray diffraction analyses of the homodimeric glycine decarboxylase (P-protein) from the cyanobacterium Synechocystis sp. PCC 6803

Abstract

Glycine decarboxylase, or P-protein, is a major enzyme that is involved in the C(1) metabolism of all organisms and in the photorespiratory pathway of plants and cyanobacteria. The protein from Synechocystis sp. PCC 6803 is a homodimer with a mass of 215 kDa. Recombinant glycine decarboxylase was expressed in Escherichia coli and purified by metal-affinity, ion-exchange and gel-filtration chromatography. Crystals of P-protein that diffracted to a resolution of 2.1 A were obtained using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected from cryocooled crystals using synchrotron radiation. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 96.30, b = 135.81, c = 179.08 A.

Figure 1

Rooted tree of glycine decarboxylase proteins constructed from a ClustalX (Larkin et al., 2007 ▶) alignment of homodimeric and heterotetrameric glycine decarboxylase enzymes. The phylogenetic tree was constructed with the program TreeView (Page, 1996 ▶). To enable the alignment of enzymes with different oligomeric compositions, subunit 2 of the heterotetrameric P-proteins was fused to the carboxy-terminus of subunit 1. The tree shows that the Synechocystis P-protein clusters more closely with eukaryotic P-proteins than any of the heterotetrameric enzymes, including the T. thermophilus enzyme. E. coli glutamate decarboxylase (NP_287662) was defined as an outlier. P-protein sequences from the following eukaryotic species and bacterial isolates were used: Synechocystis sp. PCC 6803 (P74416), Thermosynechococcus elongatus Bp-1 (NP_682393), Nostoc sp. PCC 7120 (NP_488647), Oryza sativa (AAQ24377), Arabidopsis thaliana (BAE98954), Pisum sativum (P26969), Solanum tuberosum (O49954), Flaveria anomala (O49850), Vitis vinifera (XP_002279590), Mus musculus (NP_613061), Bos taurus (XP_869239), Homo sapiens (NP_000161), Rattus norvegicus (NP_001101053), Thermus thermophilus HB-8 (subunit 1, YP_143791; subunit 2, YP_143792), Bacillus sp. B14905 (subunit 1, ZP_01725792; subunit 2, ZP_01725793), Clostridium botulinum A strain ATCC 3502 (subunit 1, YP_001253239; subunit 2, YP_001253240). The scale bar shows the distance equal to 0.1 amino-acid substitutions per sequence position.

Figure 2

12.5% SDS–PAGE of the purified Synechocystis P-protein after metal-affinity chromatography. Left lane, molecular-mass markers (kDa); right lane, P-protein.

Figure 3

Crystals of recombinant P-protein. The crystal in (a) was grown in 100 mM Tris–HCl pH 7.75, 0.25 M CsCl, 17% PEG 3350 and 10 mM 2-mercaptoethanol. The crystal in (b) was grown in 100 mM Tris–HCl pH 7.75, 0.25 M LiCl, 17% PEG 3350 and 10 mM 2-mercaptoethanol. The diffraction data were collected from a crystal grown using conditions similar those used to obtain the crystal in (a).

Figure 4

X-ray diffraction image of the apo P-protein crystal used for data collection. The diffraction data were collected on ESRF beamline ID14-4; the oscillation range was 0.5°. The edge of the detector corresponds to a resolution of 2.0 Å.