Evolution and properties of alanine racemase from Synechocystis sp. PCC6803
| Autor: | Hiroyuki Ashida, Kaho Murakami, Kenji Inagaki, Yoshihiro Sawa, Hisashi Hemmi, Yugo Iwasaki, Tohru Yoshimura |
|---|---|
| Rok vydání: | 2021 |
| Předmět: | |
| Zdroj: | The Journal of Biochemistry. 171:421-428 |
| ISSN: | 1756-2651 0021-924X |
| DOI: | 10.1093/jb/mvab155 |
| Popis: | Alanine racemase (EC 5.1.1.1) depends on pyridoxal 5′-phosphate and catalyses the interconversion between L- and D-Ala. The enzyme is responsible for the biosynthesis of D-Ala, which is an essential component of the peptidoglycan layer of bacterial cell walls. Phylogenetic analysis of alanine racemases demonstrated that the cyanobacterial enzyme diverged before the separation of gram-positive and gram-negative enzymes. This result is interesting considering that the peptidoglycans observed in cyanobacteria seem to combine the properties of those in both gram-negative and gram-positive bacteria. We cloned the putative alanine racemase gene (slr0823) of Synechocystis sp. PCC6803 in Escherichia coli cells, expressed and purified the enzyme protein and studied its enzymological properties. The enzymatic properties of the Synechocystis enzyme were similar to those of other gram-positive and gram-negative bacterial enzymes. Alignment of the amino acid sequences of alanine racemase enzymes revealed that the conserved tyrosine residue in the active centre of most of the gram-positive and gram-negative bacterial enzymes has been replaced with tryptophan in most of the cyanobacterial enzymes. We carried out the site-directed mutagenesis involving the corresponding residue of Synechocystis enzyme (W385) and revealed that the residue is involved in the substrate recognition by the enzyme. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|