A S180F substitution in D-alanine aminotransferase confers resistance to β-chloro-D-alanine in Staphylococcus aureus

Rakesh Roy; Yahani P Jayasinghe; Sasmita Panda; Merve S Zeden; Vinai C Thomas; Donald R Ronning; James P O'Gara

doi:10.1016/j.jbc.2025.110931

A S₁₈₀F substitution in D-alanine aminotransferase confers resistance to β-chloro-D-alanine in Staphylococcus aureus

J Biol Chem. 2025 Nov 11;301(12):110931. doi: 10.1016/j.jbc.2025.110931. Online ahead of print.

Authors

Rakesh Roy¹, Yahani P Jayasinghe², Sasmita Panda³, Merve S Zeden¹, Vinai C Thomas³, Donald R Ronning⁴, James P O'Gara⁵

Affiliations

¹ Microbiology, School of Biological and Chemical Sciences, University of Galway, Ireland.
² Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center Omaha, Nebraska, USA; UNMC Center for Drug Design and Innovation, University of Nebraska Medical Center Omaha, Nebraska, USA.
³ Department of Pathology, Microbiology and Immunology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
⁴ Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center Omaha, Nebraska, USA; UNMC Center for Drug Design and Innovation, University of Nebraska Medical Center Omaha, Nebraska, USA. Electronic address: don.ronning@unmc.edu.
⁵ Microbiology, School of Biological and Chemical Sciences, University of Galway, Ireland. Electronic address: jamesp.ogara@universityofgalway.ie.

PMID: 41232672
DOI: 10.1016/j.jbc.2025.110931

Abstract

β-lactam antibiotics that target the synthesis of bacterial cell wall peptidoglycan (PG) remain a cornerstone of antimicrobial chemotherapy. The activity of β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) can be enhanced by the alanine analog d-cycloserine (DCS), which blocks alanine racemase (Alr1) and d-alanine ligase (Ddl) and interferes with the supply of d-alanine for PG synthesis. Here, we report that another alanine analogue antibiotic, β-chloro-d-alanine (BCDA), also interferes with the d-alanine pathway by targeting d-alanine aminotransferase (Dat), which converts pyruvate to d-alanine. A Dat S₁₈₀F substitution, identified in a BCDA resistant MRSA mutant, resulted in reduced transaminase activity. X-ray crystallographic analysis of S. aureus Dat allowed structural modeling of the Dat-S₁₈₀F variant, which revealed an active-site loop shift that altered PLP co-factor binding. Molecular docking analysis suggests that the S₁₈₀F substitution promotes BCDA-PLP adduct dissociation via a mechanism that releases inactivated BCDA, thereby conferring resistance. These findings reveal a novel mechanism of BCDA resistance in S. aureus and the therapeutic potential of targeting Alr1 and Dat with DCS and BCDA, respectively, as part of an alternative treatment strategy for MRSA infections.

Keywords: BCDA resistance; MRSA; alanine auxotrophy; alanine metabolism; alanine racemase; crystal structure; d-alanine aminotransferase.