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Abstract

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

Original languageEnglish
Article number5313
JournalScientific Reports
Volume8
Issue number1
DOIs
Publication statusPublished - 1 Dec 2018

Fingerprint

Peptidoglycan
Gram-Positive Bacteria
Glutamine
Staphylococcus aureus
Glutaminase
Vitamin B 12
Microbial Drug Resistance
Cell Wall
Hydrogen
Catalytic Domain
Homeostasis
Magnetic Resonance Spectroscopy
Bacteria
Enzymes
Infection
Therapeutics

Keywords

  • Arginine
  • Ligases
  • Arginine biosynthesis

Cite this

@article{2f333d5cc4da48f2935c258e79f7a786,
title = "First insights of peptidoglycan amidation in Gram-positive bacteria-The high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD",
abstract = "Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 {\AA} resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.",
keywords = "Arginine, Ligases, Arginine biosynthesis",
author = "Francisco Leisico and Vieira, {Diana V.} and Figueiredo, {Teresa A.} and Micael Silva and Cabrita, {Eurico J.} and Sobral, {Rita G.} and Ludovice, {Ana Madalena} and Jos{\'e} Trinc{\~a}o and Rom{\~a}o, {Maria Jo{\~a}o} and {De Lencastre}, Herm{\'i}nia and Teresa Santos-Silva",
note = "info:eu-repo/grantAgreement/FCT/5876/147258/PT# The authors thank the Oxford Protein Production Facility, Research Complex (Harwell, Didcot, UK) for access to high-throughput facilities and assistance during the experiments; beamline staff at I02 and I04 for assistance during data collection at Diamond Light Source (Didcot, UK). DVV, TAF, FL and MS were supported by fellowships SFRH/BD/62415/2009, SFRH/BD/36843/2007, info:eu-repo/grantAgreement/FCT/5876/147258/PT PD/BD/105737/2014 and PD/BD/128202/2016 from Fundacao para a Ciencia e Tecnologia (FCT-MCTES), Portugal. This project was supported by Project PTDC/BIA-MIC/3195/2012 from FCT-MCTES, Portugal, Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER - COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), Pest-OE/BIA/UI0457/2001 (CREM) and Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of the National NMR Network (PTNMR) and are supported by Infrastructure Project No 022161 (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC).",
year = "2018",
month = "12",
day = "1",
doi = "10.1038/s41598-018-22986-3",
language = "English",
volume = "8",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",

}

TY - JOUR

T1 - First insights of peptidoglycan amidation in Gram-positive bacteria-The high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD

AU - Leisico, Francisco

AU - Vieira, Diana V.

AU - Figueiredo, Teresa A.

AU - Silva, Micael

AU - Cabrita, Eurico J.

AU - Sobral, Rita G.

AU - Ludovice, Ana Madalena

AU - Trincão, José

AU - Romão, Maria João

AU - De Lencastre, Hermínia

AU - Santos-Silva, Teresa

N1 - info:eu-repo/grantAgreement/FCT/5876/147258/PT# The authors thank the Oxford Protein Production Facility, Research Complex (Harwell, Didcot, UK) for access to high-throughput facilities and assistance during the experiments; beamline staff at I02 and I04 for assistance during data collection at Diamond Light Source (Didcot, UK). DVV, TAF, FL and MS were supported by fellowships SFRH/BD/62415/2009, SFRH/BD/36843/2007, info:eu-repo/grantAgreement/FCT/5876/147258/PT PD/BD/105737/2014 and PD/BD/128202/2016 from Fundacao para a Ciencia e Tecnologia (FCT-MCTES), Portugal. This project was supported by Project PTDC/BIA-MIC/3195/2012 from FCT-MCTES, Portugal, Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER - COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), Pest-OE/BIA/UI0457/2001 (CREM) and Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of the National NMR Network (PTNMR) and are supported by Infrastructure Project No 022161 (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC).

PY - 2018/12/1

Y1 - 2018/12/1

N2 - Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

AB - Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

KW - Arginine

KW - Ligases

KW - Arginine biosynthesis

UR - http://www.scopus.com/inward/record.url?scp=85044530342&partnerID=8YFLogxK

U2 - 10.1038/s41598-018-22986-3

DO - 10.1038/s41598-018-22986-3

M3 - Article

VL - 8

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 5313

ER -