Kinetics and Optimization of the Lysine–Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae

Journal Article · Tue May 12 00:00:00 EDT 2020 · Bioconjugate Chemistry

DOI:https://doi.org/10.1021/acs.bioconjchem.0c00163· OSTI ID:1799607

Sue, Christopher K. ^[1]; McConnell, Scott A. ^[2]; Ellis-Guardiola, Ken ^[2]; Muroski, John M. ^[2]; McAllister, Rachel A. ^[2]; Yu, Justin ^[2]; Alvarez, Ana I. ^[2]; Chang, Chungyu ^[3]; Ogorzalek Loo, Rachel R. ^[2]; Loo, Joseph A. ^[4]; Ton-That, Hung ^[3]; Clubb, Robert T. ^[5]

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1. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry. UCLA-DOE Inst. for Genomics and Proteomics; OSTI
2. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry. UCLA-DOE Inst. for Genomics and Proteomics
3. Univ. of California, Los Angeles, CA (United States). Molecular Biology Inst. Division of Oral Biology and Medicine. School of Dentistry
4. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry. Molecular Biology Inst.
5. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry. UCLA-DOE Inst. for Genomics and Proteomics. Molecular Biology Inst.

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine–isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (^CdSrtA) has been reconstituted in vitro. A mutationally activated form of ^CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (^NSpaA), enabling the labeling of target proteins that are fused to ^NSpaA. Here we present a detailed analysis of the ^CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the ^CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in ^NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (^CdSrtA^Δ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage (“lid”) that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the ^NSpaA substrate. Quantitative measurements reveal that ^CdSrtA^Δ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h^–1 and that it has apparent K_M values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its ^NSpaA and peptide substrates, respectively. ^CdSrtA^Δ is 7-fold more active than previously studied variants, labeling >90% of ^NSpaA with peptide within 6 h. The results of this study further improve the utility of ^CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.

• Research Organization: Univ. of California, Los Angeles, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: FC02-02ER63421
• OSTI ID: 1799607
• Alternate ID(s): OSTI ID: 1833089
• Journal Information: Bioconjugate Chemistry, Journal Name: Bioconjugate Chemistry Journal Issue: 6 Vol. 31; ISSN 1043-1802
• Publisher: American Chemical Society
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
catalysis
high-performance liquid chromatography
labeling
monomers
peptides and proteins