Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440

Environ Microbiol Rep. 2017 Oct;9(5):581-588. doi: 10.1111/1758-2229.12576. Epub 2017 Sep 21.

Abstract

The genome of Pseudomonas putida KT2440 contains two open reading frames (ORFs), PP_3722 and PP_5269, that encode proteins with a Pyridoxal phosphate binding motif and a high similarity to alanine racemases. Alanine racemases play a key role in the biosynthesis of D-alanine, a crucial amino acid in the peptidoglycan layer. For these ORFs, we generated single and double mutants and found that inactivation of PP_5269 resulted in D-alanine auxotrophy, while inactivation of PP_3722 did not. Furthermore, as expected, the PP_3722/PP_5269 double mutant was a strict auxotroph for D-alanine. These results indicate that PP_5269 is an alr allele and that it is the essential alanine racemase in P. putida. We observed that the PP_5269 mutant grew very slowly, while the double PP_5269/PP_3722 mutant did not grow at all. This suggests that PP_3722 may replace PP_5269 in vivo. In fact, when the ORF encoding PP_3772 was cloned into a wide host range expression vector, ORF PP_3722 successfully complemented P. putida PP_5269 mutants. We purified both proteins to homogeneity and while they exhibit similar KM values, the Vmax of PP_5269 is fourfold higher than that of PP_3722. Here, we propose that PP_5269 and PP_3722 encode functional alanine racemases and that these genes be named alr-1 and alr-2 respectively.

MeSH terms

  • Alanine Racemase / genetics*
  • Alanine Racemase / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Computational Biology / methods
  • Gene Deletion
  • Genotype
  • Mutation
  • Phenotype
  • Pseudomonas putida / enzymology*
  • Pseudomonas putida / genetics*
  • Pseudomonas putida / ultrastructure

Substances

  • Bacterial Proteins
  • Alanine Racemase