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Abstract Bacterial sortases are a family of cysteine transpeptidases in Gram‐positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase‐mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition byStaphylococcus aureusSrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif. Previous structural studies revealed that the requirement of a glycine in the binding motif may be due to potential steric hindrance of amino acids possessing a β‐carbon by W194, a tryptophan located in the β7‐β8 loop of the enzyme. Here, we measured the effect of seven single point mutants of W194 (A, D, F, G, N, S, Y) saSrtA using a FRET‐based activity assay. We found that while the LPXTG motif remains a requirement for initial proteolytic cleavage, the nucleophile specificity of our variants is altered. In particular, W194A and W194S saSrtA recognize a D‐Ala nucleophile and are able to perform ligation reactions. Notably, an LPXT(D‐Ala) peptide was not cleaved by either mutant enzyme. We hypothesize that these variants may potentially be utilized to develop an irreversible sortase‐mediated reaction. Taken together, this experiment reveals new insight into sortase specificity and possible future SML strategies.