title: Traceless splicing enabled by substrate-induced activation of the Nostoc punctiforme Npu DnaE intein after mutation of a catalytic cysteine to serine.
abstract: Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices.
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Manoj
authors.initials: M
authors.lastname: Cheriyan
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Siu-Hong
authors.initials: SH
authors.lastname: Chan
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA. Electronic address: perler@neb.com.
authors.forename: Francine
authors.initials: F
authors.lastname: Perler
mesh_terms: D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D047669:Exteins / Q000235:genetics; D047668:Inteins / Q000235:genetics*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D046937:Nostoc / Q000201:enzymology / Q000235:genetics*; D019154:Protein Splicing; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D013912:Threonine; D012694:Serine; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D000446:Aldehyde-Lyases; C100131:phospho-2-keto-3-deoxy-gluconate aldolase; D003545:Cysteine
keywords: enzyme mechanism; extein specificity; genetic selection; intein; protein engineering