Abstract
Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.
Highlights
Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce
To solve the chicken-and-egg biosynthetic puzzle for cysteine, we designed a total of four modified synthetic genes corresponding to either cysteine-free (C) or cysteine-free and methionine-deficient (CM) versions of E. coli cysE and cysM genes (Fig. 2)
The cysK-C gene was not able to restore the growth of our ΔcysKΔcysM auxotrophic E. coli strain and we subsequently focused on cysE and cysM variants
Summary
Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/ CysM) To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. It seems likely that in an earlier time polypeptides composed of a limited abiotically-derived set of amino acids might have served as biosynthetic enzymes[5] Such polypeptides could have played a role in developing and diversifying amino acid biosynthesis pathways. We focused on the biosynthesis of cysteine, an amino acid that plays an important role in metabolism, as well as modern protein structure and function. Despite the importance all of these functions suggest, cysteine has been one of the poorly identified compounds via prebiotic synthesis[11]
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