Abstract
L-Cysteine biosynthesis has been extensively analyzed in Salmonella enterica. The cysteine regulon contains the genes whose protein products are necessary to convert sulfate to sulfide, which is eventually reacted with O-acetyl-serine (OAS) to generate cysteine. The LysR type regulator, CysB, is required for activation of the cysteine regulon, and its interaction with various cys genes has been thoroughly characterized. Results from previous studies by others, suggested that OAS undergoes a spontaneous O- to N- migration to produce N-acetyl-serine (NAS), and that NAS is the true signal sensed by CysB. It was unclear, however, whether such migration occurred spontaneously in vivo or if NAS was generated enzymatically. Work reported herein characterizes a S. enterica N-acetyltransferase, OatA (formerly YjgM), which acetylates the Nα-amino group of OAS, producing N,O-diacetyl-serine (DAS) at the expense of acetyl-CoA. We isolated OatA to homogeneity and performed its initial biochemical characterization. The product of the OatA reaction was isolated by HPLC and confirmed by mass spectrometry to be DAS; OatA did not acetylate NAS, consistent with the conclusion that OatA is an N-acetyltransferase, not an O-acetyltransferase. Binding of OAS to OatA appears to be positively cooperative with the apparent K0.5 for OAS determined to be 0.74 mM, the kcat was 1.05 s-1, and the catalytic efficiency of the enzyme (kcat/K0.5) was 1.4 × 103 M-1 s-1. Size exclusion chromatography indicated that OatA was a monomer in solution. In S. enterica, overexpression of oatA led to shorter lag times on sulfate-limiting medium and that these delayed lag times were due to increased expression of the cysteine regulon, as indicated by RT-qPCR results. OatA is the first Gcn5-related N-acetyltransferase (aka GNAT) involved in the regulation of amino acid biosynthetic genes in Salmonella. On the basis of results of transcriptomics studies performed by other investigators, we hypothesize that DAS may play a role in biofilm formation in S. enterica and other bacteria.
Highlights
L-Cysteine (L-Cys) is one of the 22 proteinogenic amino acids in nature, and one of only two amino acids whose side chains contain a sulfur atom
The screen was performed using purified uncharacterized N-acetyltransferases from S. enterica (STM1857, ElaA, YafP, YhbS, YiiD, YjaB, YjgM, and YpfL). From these experiments we showed that YjgM acetylated OAS in vitro, a product we suspected to be N,O-diacetyl-serine (DAS, Figure 2)
Based on the biochemical data reported here, we suggest changing the name of the protein from YjgM to OAS acetyltransferase A or OatA, and that the yjgM gene be referred to as oatA
Summary
L-Cysteine (L-Cys) is one of the 22 proteinogenic amino acids in nature, and one of only two amino acids whose side chains contain a sulfur atom. The sulfhydryl group of L-Cys is critical to maintaining redox homeostasis in cells from all domains of life (Hernandez et al, 2015; Hasan et al, 2016; Zhang et al, 2016; Cao et al, 2017; Liu et al, 2018; Tang et al, 2018). The biosynthesis of this important amino acid has been extensively studied (Kredich, 2008). Previous literature has characterized binding of CysB to the promoter regions of cysK [encodes O-acetylserine (thiol)-lyase, EC 2.5.1.47], cysJIH (encode sulfite reductase, EC 1.8.7.1), cysP (encodes thiosulfatebinding protein), and to cysB (encodes a LysR regulator) (Monroe et al, 1990; Hryniewicz and Kredich, 1991, 1994; Ostrowski and Kredich, 1991)
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