D-GLYCERATE 3-KINASE, the last unknown enzyme in the photorespiratory cycle in Arabidopsis, belongs to a novel kinase family

Abstract

D-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C2 cycle, an indispensable ancillary metabolic pathway to the photosynthetic C3 cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C2 cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO2, providing direct evidence of the obligatory nature of the ultimate step of the C2 cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.

Figure 1.

The C₂ and C₃ Cycles of Photosynthetic Carbon Metabolism Are Tied up with Each Other via the Oxygenation of RubP and the Phosphorylation of Glycerate.

Figure 2.

Purification of GLYK from Arabidopsis Leaves. (A) Elution profile during DEAE ion-exchange chromatography. Circles indicate GLYK activity, and triangles show protein concentration. (B) SDS-PAGE of pooled fractions with GK activity. (C) Final affinity chromatography on Matrex Green A affinity yields electrophoretically homogeneous enzyme. (D) SDS-PAGE of 5.0 μg of purified enzyme.

Figure 3.

Arabidopsis GLYK Is Encoded by the Gene At1g80380. (A) Complete amino acid sequence of Arabidopsis GLYK with experimentally determined partial amino acid sequences shown in bold. (B) GLYK is localized at the very end of the right arm of chromosome 1 and comprises 11 exons (∼3500 bp). T-DNA insertion sites in the knockout lines are marked with triangles. UTR, untranslated region. (C) The protein includes a 118–amino acid chloroplastic transit peptide (cTP), an ATP/GTP binding site motif A (P-loop), and the motifs GLYK-1 and GLYK-2, which are specific for the GLYK protein family.

Figure 4.

The Recombinant Protein Encoded by At1g80380 Shows GLYK Activity. (A) GLYK activities measured in E. coli BL-21 lysate harboring the empty and the recombinant vector, respectively, or with the purified recombinant protein. (B) SDS-PAGE of recombinant GLYK purified by affinity chromatography on Matrex Green A.

Figure 5.

GLYK Is Indispensable for Normal Plant Development and Growth, but This Absolute Requirement Is Relaxed under Elevated CO₂. (A) GLYK knockout mutants do not survive in normal air but recover at elevated CO₂. From left to right: Arabidopsis wild type and Atglyk1-1 and Atglyk1-2 grown in normal air in comparison with Atglyk1-1 grown under 1200 μL L⁻¹ CO₂. All plants are 2 weeks after germination. The mutants do not develop primary leaves in normal air but do so within 4 to 5 d after transfer to elevated CO₂. (B) The homozygous knockout plants do not show residual GLYK mRNA. The 40S ribosomal S16 protein mRNA was used as internal control in the RT-PCR analysis.

Figure 6.

At1g80380 Is the Exclusive Source of GLYK Activity in Arabidopsis Leaves. (A) GLYK activity in wild-type and knockout mutants. Residual activity is close to zero and within the range of experimental error in Atglyk1-1 and Atglyk1-2. (B) Leaf glycerate content is greatly elevated, whereas the amounts of hydroxypyruvate and Ser are only approximately fivefold enhanced. Metabolite content is depicted in relative units with the respective wild-type values set to one. Bars represent standard errors of the mean.

Figure 7.

GLYK Represents a Novel Kinase Family Phylogenetically Distinct from Other GK Proteins. The neighbor-joining tree was constructed from a ClustalX (Thompson et al., 1997) alignment of GLYK, GK, and PRK amino acid sequences with the program TREECON 1.3b (Van de Peer and De Wachter, 1994) using a Poisson correction for the calculation of the distance matrix. Bootstrap analysis with 1000 replicates (results shown as percentage values) confirmed the tree topology at all nodes and the differentiation into four unrelated classes. Sequences used for calculations are as follows: E. coli GK1 (E. coli glxK, P77364), E. coli GK2 (E. coli garK, P23524), E. coli GK3 (E. coli gcxK putative GK, AAL61903), Erwinia GK (E. carotovora garK, CAG76470), Klebsiella GK (K. pneumoniae glxK, BAD14998), Synechocystis GK (Synechocystis sp PCC 6803 hypothetical protein, P73408), mouse GK (Mus musculus transcript, AAH25834), human GK (Homo sapiens GLYCTK1, AAP41923), Silicibacter GK (Silicibacter sp TM 1040 putative GK, ZP_00336015), Desulfovibrio GK (D. desulfuricans putative GK, ZP00129227), Thermotoga GK (T. maritima putative GK, AAD36652), Anabaena GLYK (A. variabilis predicted kinase, ZP_00161317), Nostoc GLYK (Nostoc sp PCC 7120 hypothetical protein, NP_486913), S. cerevisiae GLYK (S. cerevisiae ATP binding protein, NP011721), S. pombe GLYK (S. pombe ATP binding protein, P78883), rice GLYK (Oryza sativa PRK/UK-like protein, BAD73764, Os01g48990), Arabidopsis GLYK (At1g80380, this article), Synechocystis PRK (Synechocystis sp PCC 6803 PRK, AAA27293), Anabaena PRK (A. variabilis PRK/UK, AD2321), Arabidopsis PRK (P25697), and Chlamydomonas PRK (C. reinhardtii PRK, P19824).