DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Structure and Mechanism of LcpA, a Phosphotransferase That Mediates Glycosylation of a Gram-Positive Bacterial Cell Wall-Anchored Protein

Journal Article · · mBio (Online)
 [1];  [2];  [1];  [2];  [2];  [2]; ORCiD logo [3];  [3]
  1. Univ. of Texas Health Science Center, Houston, TX (United States)
  2. Univ. of California, Los Angeles, CA (United States). Inst. of Genomics and Proteomics and Dept. of Chemistry and Biochemistry
  3. Univ. of Texas Health Science Center, Houston, TX (United States); Univ. of California, Los Angeles, CA (United States)

The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-β-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM. Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms. In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
Grant/Contract Number:
FC02-02ER63421; AI52217; AI121360; DE017382; DE025015
OSTI ID:
1494967
Alternate ID(s):
OSTI ID: 1499711
Journal Information:
mBio (Online), Vol. 10, Issue 1; ISSN 2150-7511
Publisher:
American Society for MicrobiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 18 works
Citation information provided by
Web of Science

References (47)

A widespread family of bacterial cell wall assembly proteins: Bacterial cell wall assembly proteins journal September 2011
Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris journal June 2017
Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions journal March 2008
Structure of the collagen-binding domain from a Staphylococcus aureus adhesin journal October 1997
Lcp1 Is a Phosphotransferase Responsible for Ligating Arabinogalactan to Peptidoglycan in Mycobacterium tuberculosis journal August 2016
NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy journal December 2014
A short history of SHELX journal December 2007
In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins journal February 2017
Bacterial coaggregation: an integral process in the development of multi-species biofilms journal February 2003
High-Throughput Thermal Scanning: A General, Rapid Dye-Binding Thermal Shift Screen for Protein Engineering journal March 2009
Lethality of sortase depletion in A ctinomyces oris caused by excessive membrane accumulation of a surface glycoprotein journal September 2014
A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris journal May 2016
A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris journal July 2015
Features and development of Coot journal March 2010
Substrate Preferences Establish the Order of Cell Wall Assembly in Staphylococcus aureus journal February 2018
Biogenesis of the Gram-positive bacterial cell envelope journal December 2016
B. subtilis LytR-CpsA-Psr Enzymes Transfer Wall Teichoic Acids from Authentic Lipid-Linked Substrates to Mature Peptidoglycan In Vitro journal December 2017
Allelic Exchange in Actinomyces oris with mCherry Fluorescence Counterselection journal July 2010
PHENIX: a comprehensive Python-based system for macromolecular structure solution journal January 2010
Structural Characterization of the Lactobacillus Plantarum FlmC Protein Involved in Biofilm Formation journal September 2018
Evidence for a Catalytically and Kinetically Competent Enzyme–Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase journal July 2014
Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria journal December 2015
Enzymatic Assembly of Overlapping DNA Fragments book May 2011
Wall Teichoic Acids of Gram-Positive Bacteria journal September 2013
Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega journal January 2011
Attachment of Capsular Polysaccharide to the Cell Wall in Streptococcus pneumoniae journal June 2012
An expression vector tailored for large-scale, high-throughput purification of recombinant proteins journal June 2006
Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm journal November 1999
NMRPipe: A multidimensional spectral processing system based on UNIX pipes journal November 1995
LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly journal November 2014
Protein glycosylation in bacteria: sweeter than ever journal October 2010
Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris journal March 2017
Characterization of the Streptococcus adjacens group antigen structure. journal January 1992
Integration, scaling, space-group assignment and post-refinement journal January 2010
Release of protein A from the cell wall of Staphylococcus aureus journal January 2014
Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum journal August 2016
Molecular Analysis of the tagF Gene, Encoding CDP-Glycerol:Poly(glycerophosphate) Glycerophosphotransferase of Staphylococcus epidermidis ATCC 14990 journal February 2000
The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes journal April 2014
Streptococcus pneumoniae capsular polysaccharide is linked to peptidoglycan via a direct glycosidic bond to β-D- N -acetylglucosamine journal May 2017
The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates journal September 2014
Lipoteichoic Acid Synthesis and Function in Gram-Positive Bacteria journal September 2014
Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER journal March 2012
Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23 journal December 1986
PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome journal December 2019
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation journal February 2021
Multi-functionality of a tryptophan residue conserved in substrate-binding groove of GH19 chitinases journal January 2021
PHENIX: a comprehensive Python-based system for macromolecular structure solution. text January 2010

Cited By (1)

Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes journal August 2019