Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence ofCorynebacterium glutamicum

Abstract

Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with L-glutamate, L-aspartate, and L-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) L-glutamate > 2-aminobutyrate > L-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes L-alanine to aminate 2-oxo-isovalerate, the L-valine precursor, and 2-oxo-butyrate. A second AT active with the L-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for L-phenylalanine synthesis. We also assessed the activity of two PLPcontaining cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

Positive transcriptional control of the pyridoxal phosphate biosynthesis genes pdxST by the MocR-type regulator PdxR of Corynebacterium glutamicum ATCC 13032

“…However, the amino acid sequence alignment of PLP attachment sites from the actinobacterial PdxR-like proteins revealed the lack of the highly conserved lysine residue that is generally used for the covalent binding of the PLP group (Sigrist et al, 2010). Replacement of this essential lysine residue by serine, threonine or histidine may result in the loss of an enzymic function, which is consistent with a previous functional analysis in C. glutamicum, as aminotransferase activity was not detectable in enzyme assays with the purified PdxR protein (Marienhagen et al, 2005). This result is in marked contrast with observations made with the regulatory protein GabR from Bacillus subtilis, a MocRtype transcription regulator that positively controls the utilization of c-aminobutyric acid and represses its own expression (Belitsky & Sonenshein, 2002).…”

“…This result suggests that the aminotransferase domain of the actinobacterial PdxR-like proteins is probably unable to bind PLP covalently. At least in the case of C. glutamicum ATCC 13032, this indication fits the previous observation that no aminotransferase activity was detectable in enzyme assays with purified PdxR protein (Marienhagen et al, 2005).…”

Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate

“…It is very likely that FdhD from C. glutamicum ATCC 13032 also acts as a sulfur transferase between a desulfurase and FdhF and therefore is essential for the formation of an active FDH. Several homologues of IscS from E. coli can also be found in the genomes mentioned above and have also been described in C. glutamicum ATCC 13032 (cg1214, cg1388 and cg1761) (see Schaffer et al, 2001;Marienhagen et al, 2005;Teramoto et al, 2010).…”

Isolation of a Mutant Auxotrophic forL-Alanine and Identification of Three Major Aminotransferases That SynthesizeL-Alanine inEscherichia coli

“…Isolation of an alanine auxotroph by random mutation Since the uncharacterized gene product, YfbQ, of E. coli appeared to possess high homology (61% identity) with the recently identified alanine aminotransferase, AlaT, of C. glutamicum 14) and we found that histidine-tagged YfbQ had alanine aminotransferase activity (Tachaapaikoon et al, unpublished results), we predicted that a double-knockout mutant deficient in both the avtA and the yfbQ gene might display alanine auxotrophy if their gene products play a major role in L-alanine formation in vivo. To evaluate this hypothesis, we constructed double knockout mutant HYE021 lacking both avtA and yfbQ.…”

“…php?spid=Cglu ATCC13032&ftid=2921) is involved in L-alanine synthesis in this bacterium (Lim et al, unpublished results), which coincidently appears to be the same product of the alaT gene of C. glutamicum. 14) The fact that AlaT of C. glutamicum has high homology (61% identity) with an uncharacterized homolog, YfbQ, in the E. coli genome, prompted us to elucidate the contribution of YfbQ to L-alanine synthesis.…”