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Biochem Biophys Res Commun. 2017 Jan 29;483(1):122-128. doi: 10.1016/j.bbrc.2016.12.183. Epub 2016 Dec 29.

Structural and functional characterization of the alanine racemase from Streptomyces coelicolor A3(2).

Author information

1
Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
2
Microbial Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
3
Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. Electronic address: m.ubbink@chem.leidenuniv.nl.
4
Microbial Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands. Electronic address: g.wezel@biology.leidenuniv.nl.
5
Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. Electronic address: raj@chem.leidenuniv.nl.

Abstract

The conversion of l-alanine (L-Ala) into d-alanine (D-Ala) in bacteria is performed by pyridoxal phosphate-dependent enzymes called alanine racemases. D-Ala is an essential component of the bacterial peptidoglycan and hence required for survival. The Gram-positive bacterium Streptomyces coelicolor has at least one alanine racemase encoded by alr. Here, we describe an alr deletion mutant of S. coelicolor which depends on D-Ala for growth and shows increased sensitivity to the antibiotic d-cycloserine (DCS). The crystal structure of the alanine racemase (Alr) was solved with and without the inhibitors DCS or propionate, at 1.64 Å and 1.51 Å resolution, respectively. The crystal structures revealed that Alr is a homodimer with residues from both monomers contributing to the active site. The dimeric state of the enzyme in solution was confirmed by gel filtration chromatography, with and without L-Ala or d-cycloserine. The activity of the enzyme was 66 ± 3 U mg-1 for the racemization of L- to D-Ala, and 104 ± 7 U mg-1 for the opposite direction. Comparison of Alr from S. coelicolor with orthologous enzymes from other bacteria, including the closely related d-cycloserine-resistant Alr from S. lavendulae, strongly suggests that structural features such as the hinge angle or the surface area between the monomers do not contribute to d-cycloserine resistance, and the molecular basis for resistance therefore remains elusive.

KEYWORDS:

Actinobacteria; Alanine racemase; Antibiotic resistance; Peptidoglycan; X-ray crystallography

PMID:
28042035
DOI:
10.1016/j.bbrc.2016.12.183
[Indexed for MEDLINE]

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