Premium
Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440
Author(s) -
Duque Estrella,
Daddaoua Abdelali,
Cordero Baldo F.,
De la Torre Jesús,
Antonia MolinaHenares Maria,
Ramos JuanLuis
Publication year - 2017
Publication title -
environmental microbiology reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.229
H-Index - 69
ISSN - 1758-2229
DOI - 10.1111/1758-2229.12576
Subject(s) - orfs , biology , mutant , biochemistry , pseudomonas putida , open reading frame , alanine , genetics , microbiology and biotechnology , peptide sequence , gene , amino acid
Summary 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 K M values, the V max 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.