Abstract
The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors.
Highlights
Dipeptides constitute a promising substance class and are often more than the sum of their parts
The selection of important positions for substrate specificity was based on the structure of RizA, a sequence alignment of RizA with BacD, Bl-L-amino acid ligases (LALs) [23], RsLAL [22], and TabS [27], and data from the literature on the substrate specificity and engineering of these LALs
Since the published structure of RizA does not contain a substrate, a structural alignment with BacD containing a substrate analog of the Ala-Phe dipeptide was used to approximate substrate binding as has been previously done in the original publication on the RizA structure [28] and for the engineering of Bl-LAL [12]
Summary
Dipeptides constitute a promising substance class and are often more than the sum of their parts. The discovery of the first LAL YwfE ( called BacD) from Bacillus subtilis was published in 2005 [20] It synthesized a large variety of different dipeptides and generally preferred smaller amino acids like alanine as the N-terminal and larger amino acids like phenylalanine as its C-terminal substrate. Another recent engineering study improved the production of Pro-Gly by the LAL TabS from Pseudomonas syringae NBRC14081 [24] Among these enzymes with different substrate specificities, RizA from Bacillus subtilis NBRC3134 stands out due to its high N-terminal specificity accepting only arginine, but relaxed C-terminal specificity accepting every amino acid except proline [25]. Since a crystallographic structure of RizA has been published [28], a mutagenesis study of the substrate binding pocket of RizA was conceived to increase the production of several bioactive dipeptides and to gain knowledge about the substrate specificity of LALs for future engineering endeavors
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