Appl Microbiol Biotechnol (2002) 60:437–441 DOI 10.1007/s00253-002-1161-y SHORT CONTRIBUTION P. Peters-Wendisch · R. Netzer · L. Eggeling · H. Sahm 3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum : the C-terminal domain is not essential for activity but is required for inhibition by l-serine Received: 20 June 2002 / Revised: 23 September 2002 / Accepted: 27 September 2002 / Published online: 6 November 2002  Springer-Verlag 2002 Abstract The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized. It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa. The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide. Overexpression of serA in C. glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of l-serine. A set of muteins that were progressively truncated at the C-terminal end was constructed. When overexpressed, mutein SerAD197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to l-serine. Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerAD197 constitutes a dimer. Thus, the specific regula- tory features of C. glutamicum PGDH are due to the C- terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme. Introduction The amino acid l-serine is a central cellular intermediate, which serves not only as a building block for protein synthesis but also as a precursor for the synthesis of, e.g., glycine, cysteine, tryptophan, phospholipids, purines and C1 units. Therefore, although about 15% of the glycolytic flux is directed into the l-serine biosynthetic pathway, only 6% of it leads to incorporation of l-serine into protein (McKitrick and Pizer 1980). In many bacteria, vertebrates and plants (Saski and Pizer 1975; McKitrick and Pizer 1980; Achouri et al. 1997; Ho and Saito 2001), l-serine is synthesized via a “phosphorylated” pathway with the first reaction initiated by the enzyme 3-phosphoglycerate dehydrogenase (PGDH) oxidizing 3-phosphoglycerate to phosphohydroxypyruvate. In Es- cherichia coli, PGDH is V max -type allosterically inhibited by l-serine (Grant et al. 1996). A specific inhibition by l- serine was also observed for PGDH from Bacillus subtilis (Saski and Pizer 1975). However, inhibition of the B. subtilis enzyme by l-serine exhibits an unusual time dependence, requiring several minutes of incubation before a constant level of inhibition is achieved. Thus, the two bacterial PGDHs characterized to date are regulated by l-serine in different ways. In contrast, PGDH assayed from vertebrate tissues and Arabidopsis thaliana are not regulated by l-serine (Achouri et al. 1997; Ho and Saito 2001). PGDH proteins, encoded by the gene serA, belong to a family of d-isomer 2-hydroxyacid dehydrogenases. The primary structure of SerA polypeptides is characterized by two highly conserved d-2-hydroxyacid binding motifs in their N-terminal part (Grant 1989). Analysis of the 3- dimensional (3D) structure of E. coli PGDH revealed a homotetramer with four active sites and four effector- binding sites. Each subunit contains three distinct glob- ular domains referred to as substrate binding and nucle- otide binding domains – containing the characteristic d-2- hydroxyacid motifs– and the regulatory domain, contain- ing a small ligand binding motif (Schuller et al. 1995; Chipman and Shaanan 2001). Similarly, PGDH from B. subtilis, rat liver and plants are most likely homotetramers (Saski and Pizer 1975; Achouri et al. 1997; Ho et al. 1999). Due to the high sequence identity of these enzymes over a long N-terminal part with the E. coli enzyme, these proteins are expected to share a very similar 3D structure with that of PGDH from E. coli (Schuller et al. 1995). Our aim was to study the PGDH from Corynebacte- rium glutamicum. This bacterium is used for the large scale synthesis of l-glutamate and l-lysine (Eggeling and Sahm 1999) and serves as a model organism to modulate fluxes of a variety of amino acid biosynthetic pathways. P. Peters-Wendisch ( ) ) · R. Netzer · L. Eggeling · H. Sahm Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52428 Jülich, Germany e-mail: p.wendisch@fz-juelich.de Tel.: +49-2461-615430 Fax: +49-2461-612710