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Biochemical Characterization of Pyranose Oxidase from Streptomyces canus-Towards a Better Understanding of Pyranose Oxidase Homologues in Bacteria
BOKU Univ Nat Resources & Life Sci, Dept Food Sci & Technol, Lab Food Biotechnol, A-1180 Vienna, Austria.;BOKU Univ Nat Resources & Life Sci, Doctoral Programme BioToP Biomol Technol Prot, A-1180 Vienna, Austria..ORCID iD: 0000-0001-9056-1634
BOKU Univ Nat Resources & Life Sci, Dept Food Sci & Technol, Lab Food Biotechnol, A-1180 Vienna, Austria..ORCID iD: 0000-0002-8889-2343
BOKU Univ Nat Resources & Life Sci, Dept Food Sci & Technol, Lab Food Biotechnol, A-1180 Vienna, Austria..
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0003-2875-0156
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2022 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 21, article id 13595Article in journal (Refereed) Published
Abstract [en]

Pyranose oxidase (POx, glucose 2-oxidase; EC 1.1.3.10, pyranose:oxygen 2-oxidoreductase) is an FAD-dependent oxidoreductase and a member of the auxiliary activity (AA) enzymes (subfamily AA3_4) in the CAZy database. Despite the general interest in fungal POxs, only a few bacterial POxs have been studied so far. Here, we report the biochemical characterization of a POx from Streptomyces canus (ScPOx), the sequence of which is positioned in a separate, hitherto unexplored clade of the POx phylogenetic tree. Kinetic analyses revealed that ScPOx uses monosaccharide sugars (such as d-glucose, d-xylose, d-galactose) as its electron-donor substrates, albeit with low catalytic efficiencies. Interestingly, various C- and O-glycosides (such as puerarin) were oxidized by ScPOx as well. Some of these glycosides are characteristic substrates for the recently described FAD-dependent C-glycoside 3-oxidase from Microbacterium trichothecenolyticum. Here, we show that FAD-dependent C-glycoside 3-oxidases and pyranose oxidases are enzymes belonging to the same sequence space.

Place, publisher, year, edition, pages
MDPI AG , 2022. Vol. 23, no 21, article id 13595
Keywords [en]
pyranose oxidase, glycosides, kinetics, structure, characterization, bacterial lignocellulose degradation, CAZy AA3
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-322008DOI: 10.3390/ijms232113595ISI: 000881157400001PubMedID: 36362382Scopus ID: 2-s2.0-85141636099OAI: oai:DiVA.org:kth-322008DiVA, id: diva2:1714635
Note

QC 20221130

Available from: 2022-11-30 Created: 2022-11-30 Last updated: 2023-11-10Bibliographically approved
In thesis
1. Symbiotic and pathogenic factors in plant-microbe interaction: Structural basis of C-glycoside metabolism and lipoprotein transport in bacteria
Open this publication in new window or tab >>Symbiotic and pathogenic factors in plant-microbe interaction: Structural basis of C-glycoside metabolism and lipoprotein transport in bacteria
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The communication between plants and bacteria involves a complex chemical signaling network that mediates responses to various biotic and abiotic stresses, as well as establishing symbiotic relationships between different organisms.

The first part of the thesis focuses on a mechanism for symbiotic communication between plants and bacteria and more specifically on how C-glycosylated aromatic polyketide compounds produced by plants can be used as a mechanism for plants to communicate with beneficial bacteria. In their glycosylated form, these compounds are substrates for symbiotic bacteria, which in return deglycosylate them and release the sugar-free part, the active aglycone. The aglycone can then mediate several functions beneficial to the plant, for example facilitating nitrogen fixation or acting as an antibacterial agent against plant pathogens.

Results from studies covered in the thesis show that the soil bacteria Deinococcus aerius, Streptomyces canus and Microbacterium testaceum produce enzymes that can cleave the carbon-carbon bond between the sugar and the aglycone in C-glycosyl compounds. Deglycosylation first requires oxidation of the sugar by an oxidoreductase, after which the carbon-carbon bond can be cleaved by a C-glycosyl deglycosidase (CGD). Biochemical and structural characterization as well as results from phylogenetic analyzes of the amino-acid sequences of CGD enzymes provided new knowledge about these relatively unexplored enzymatic processes, as well as increased insight into how C-glycosylated aromatic polyketides participate in the interaction between plant and bacteria.

The second part of the thesis explores pathogenic interactions between plants and bacteria. The virulence of pathogenic bacteria is dependent on lipoproteins that are attached to the bacteria's outer membrane and that have a decisive role for the bacteria's survival and pathogenicity. The localization of lipoproteins takes place through a process abbreviated Lol. The Lol system of the notorious plant pathogen Xanthomonas campestris was studied to better understand the underlying molecular mechanisms of the localization system, which could eventually open new ways to combat the bacterium. Biochemical, structural, and phylogenetic techniques were used also in this project.

Taken together, the results contributed several new discoveries. For the first time, a physical complex between the two proteins responsible for transporting lipoproteins could be determined and their mutual interactions studied. Furthermore, sequence analyses challenge the generally accepted model of how lipoproteins are released from the bacterial inner membrane before being transported to the outer membrane. 

According to the standard model based on Escherichia coli, lipoproteins are extracted from the inner membrane by a membrane protein that belongs to the ABC-transporter family and whose structure forms an asymmetric heterodimer (LolCDE). However, our bioinformatic analysis show that most of these ABC transporters, including X. campestris, are likely to be homodimers and that Escherichia coli is the exception rather than the rule. The difference between an asymmetric and symmetric ABC transporter also has implications for several hypotheses about how these proteins function. Heterologous production of the X. campestris ABC transporter confirmed that the protein is a homodimer.

Abstract [sv]

Kommunikationen mellan växter och bakterier omfattar ett komplext kemiskt signaleringsnätverk som förmedlar svar på olika biotiska och abiotiska påfrestningar, samt etablerar symbiotiska relationer mellan olika organismer.

Den första delen av avhandlingen fokuserar på en mekanism för symbiotisk kommunikation mellan växter och bakterier och mer specifikt på hur C-glykosylerade aromatiska polyketidföreningar producerade av växter kan utnyttjas som en mekanism för växter att kommunicrea med goda bakterier. I sin glykosylerade form utgör dessa föreningar substrat for symbiotiska bakterier som i gengäld deglykosylerar dem och frigör den sockerfria delen, det aktiva aglykonet. Aglykonet kan sedan förmedla ett flertal för växten fördelaktiga funktioner, till exempel underlättande av kvävefixering eller fungera som ett antibakteriellt medel mot växtpatogener.

I avhandlingen visas att jordbakterierna Deinococcus aerius, Streptomyces canus och Microbacterium testaceum producerar enzymer som ansvarar för klyning av kol-kol-bindingen mellan sockret och aglykonet i C-glykosylföreningar. Deglykosylering kräver först oxidation av sockret med hjälp av ett oxidoreduktas, varefter kol-kol-bindningen kan klyvas av ett C-glykosyldeglykosidas (CGD). Biokemisk och strukturell karakterisering samt resultat från fylogenetiska analyser av CGD-enzymers aminosyra-sekvenser ger ny kunskap om dessa relativt outforskade enzymatiska processer, samt ökad insikt om hur C-glykosylerade aromatiska polyketider deltar i samspelet mellan växt och bakterie.

Den andra delen av avhandlingen utforskar patogena interaktioner mellan växter och bakterier. Patogena bakteriers virulens är beroende av lipoproteiner som är fästa i bakteriens yttermembran och som har en avgörande roll för bakteriens överlevnad och patogenicitet. Lokaliseringen av lipoproteier sker genom en process som förkortas Lol. Lol-systemet hos den problematiska växtpatogenen Xanthomonas campestris studerades för att bättre förstå de underliggande molekylära mekanismerna hos lokaliseringssystemet, vilket på sikt kan öppna upp för nya sätt att bekämpa bakterien. Även i detta projekt användes biokemiska, strukturella och fylogenetiska tekniker.

Sammantaget bidrog resultaten med flera nya upptäckter. För första gången kunde ett fysiskt komplex mellan de två proteinerna som ansvarar för transport av lipoproteiner bestämmas och deras inbördes interaktioner studeras. Vidare bidrog sekevensanalyser till ifrågasättande av den allmänt vedertagna modellen för hur lipoproteiner frigörs från bakteriens innermembran innan den transporteras till yttermembranet. 

Enligt standardmodellen baserad på Escherichia coli extraheras lipoproteiner från innermembranet av ett membranprotein som tillhör ABC-transportörfamiljen och vars struktur bildar en asymmetrisk heterodimer. Våra bioinformatikstudie visade dock att merparten av dessa ABC-transportörer, inklusive den i X. campestris, mest troligt är homodimerer och att Escherichia coli är ett undantag snarare än en regel. Skillnaden mellan en asymmetrisk och symmetrisk ABC-transportör får även konsekvenser för flera hypoteser om hur dessa proteiner fungerar. Heterolog produktion av ABC-transportören från X. campestris bekräftade att proteinet är en homodimer.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2023. p. 96
Series
TRITA-CBH-FOU ; 2023:53
Keywords
Plant-Bacteria Interaction, Symbiotic Communication, C-Glycosylated Compounds, Enzymatic Deglycosylation, Pathogenic Interactions, Lipoprotein Localization, Lol System
National Category
Natural Sciences Biochemistry and Molecular Biology Structural Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-339434 (URN)978-91-8040-755-7 (ISBN)
Public defence
2023-12-08, Kollegiesalen, Brinellvägen 8, https://kth-se.zoom.us/j/64106056688, Stockholm, Sweden, 13:00 (English)
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Note

QC 2023-11-10

Available from: 2023-11-10 Created: 2023-11-10 Last updated: 2023-12-04Bibliographically approved

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Furlanetto, ValentinaDivne, Christina

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