title: Structure and specificity of a new class of Ca2+-independent housekeeping sortase fromStreptomyces avermitilisprovide insights into its non-canonical substrate preference.
abstract: Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) fromStaphylococcus aureuscleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occursin vitroas well asin celluloin the presence of Ca2+and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+requirement (particularly forin celluloapplications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles inStreptomyces avermitilisBiochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.
authors.affiliation: From the Department of Physics, Indian Institute of Science, Bangalore 560012, and.
authors.forename: Sreetama
authors.initials: S
authors.lastname: Das
authors.affiliation: the National Institute of Immunology, Delhi 110067, India.
authors.forename: Vijaykumar S
authors.initials: VS
authors.lastname: Pawale
authors.affiliation: From the Department of Physics, Indian Institute of Science, Bangalore 560012, and.
authors.forename: Venkatareddy
authors.initials: V
authors.lastname: Dadireddy
authors.affiliation: the National Institute of Immunology, Delhi 110067, India.
authors.forename: Avinash Kumar
authors.initials: AK
authors.lastname: Singh
authors.affiliation: From the Department of Physics, Indian Institute of Science, Bangalore 560012, and ramak@physics.iisc.ernet.in.
authors.forename: Suryanarayanarao
authors.initials: S
authors.lastname: Ramakumar
authors.affiliation: the National Institute of Immunology, Delhi 110067, India rproy@nii.ac.in.
authors.forename: Rajendra P
authors.initials: RP
authors.lastname: Roy
mesh_terms: D020816:Amino Acid Motifs; D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D016680:Genome, Bacterial; D010455:Peptides / Q000737:chemistry; D010458:Peptidyl Transferases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D011994:Recombinant Proteins; C509824:sortase E; D019881:Aminoacyltransferases; D010458:Peptidyl Transferases; D003546:Cysteine Endopeptidases; D002118:Calcium
keywords: Streptomyces; crystal structure; enzyme catalysis; peptide; peptides; sortase A; substrate specificity; transpeptidase