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Vitreoscilla Haemoglobin - Peroxidase Activity and Metabolic Aspects

Kvist, Malin LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Funktionen hos hemoglobin från ryggradslösa organismer är mer varierande än motsvarande för ryggradsdjur, vilket möjligen speglar de mer mångfasetterade behoven inom den förra gruppen. Hemoglobin har strukturella likheter med andra hemproteiner/enzymer och uppvisar även överlappande enzymaktiviteter, med funktioner utöver transport och lagring av syre.



Under syrefattiga förhållanden producerar den aeroba bakterien Vitresocilla ett hemoglobin, så kallat VHb. Vitreoscilla antas använda VHb som en strategi för att underlätta transport av syre till andningskedjan, och på så vis kunna bibehålla tillväxt genom respiration. Vid uttryck i olika värdorganismer har VHb visat sig ha... (More)
Popular Abstract in Swedish

Funktionen hos hemoglobin från ryggradslösa organismer är mer varierande än motsvarande för ryggradsdjur, vilket möjligen speglar de mer mångfasetterade behoven inom den förra gruppen. Hemoglobin har strukturella likheter med andra hemproteiner/enzymer och uppvisar även överlappande enzymaktiviteter, med funktioner utöver transport och lagring av syre.



Under syrefattiga förhållanden producerar den aeroba bakterien Vitresocilla ett hemoglobin, så kallat VHb. Vitreoscilla antas använda VHb som en strategi för att underlätta transport av syre till andningskedjan, och på så vis kunna bibehålla tillväxt genom respiration. Vid uttryck i olika värdorganismer har VHb visat sig ha positiva effekter på bland annat tillväxten. Dessutom visar nyligen presenterade studier att VHb kan bidra till att öka tåligheten mot oxidativ- och nitrosativ stress. Trots omfattande studier är emellertid den biologiska funktionen hos VHb inte klarlagd.



Denna avhandling beskriver resultaten från två tillvägagångssätt, vilka syftar till att öka kunskapen om VHbs potentiella funktioner och användbarhet inom olika biotekniska tillämpningar. Dessa två strategier omfattar dels studier av VHbs förmåga att fungera som ett peroxidas, och dels effekten av VHb-uttryck på tillväxt och metabolism i aeroba odlingar av Lactococcus lactis och saltstressade Escherichia coli-kulturer. Våra resultat visar för första gången att VHb har en peroxidasaktivitet jämförbar med andra peroxidaser. Dessutom visar en undersökning av olika genmodifierade VHb-varianter att den katalytiska aktiviteten kan moduleras genom lokala förändringar i den proximala hemfickan, och att detta kan ske utan övergripande förändringar av proteinstrukturen. Resultaten från denna studie skulle kunna utnyttjas i olika biotekniska applikationer, genom att anpassa aktiviteten hos VHb till det aktuella användningsområdet.



Studien av VHb i L. lactis beskriver det hittills outforskade uttrycket av hemoglobin i en värdcell som saknar fullständig respirationskapacitet. Våra resultat visar att L. lactis som odlas i aeroba kulturer med tillsats av hemin och som uttrycker biologiskt aktivt VHb når högre slutlig celldensitet än kontrollceller som saknar VHb. Uttryck av VHb och en genmodifierad variant, dubbel-VHb (dVHb), i E. coli celler utsatta för höga koncentrationer av NaCl (upp till 1,1 M) visade att den slutliga celldensiteten var högre för dVHb-celler än för kontrollceller som saknar VHb. Dessutom tyder våra resultat på att dVHb-celler uppvisar en lägre grad av oxidativ stress till följd av den höga salthalten. (Less)
Abstract
The function of non-vertebrate haemoglobins (Hbs) appears more diverse than that of vertebrate Hbs, possibly reflecting the various demands to be met among their hosts. Sharing structural features with other haem proteins/enzymes, Hbs display a qualitative overlap in activities with functions extending beyond oxygen transport/storage.



When subjected to oxygen deficient conditions, the obligate aerobic bacterium Vitreoscilla synthesises a homodimeric haemoglobin, VHb, envisaged to sustain respiratory growth by facilitating oxygen-delivery to the respiratory apparatus. Heterologously expressed in various host organisms, VHb has shown to exert positive effects on growth and product formation. Furthermore, results from... (More)
The function of non-vertebrate haemoglobins (Hbs) appears more diverse than that of vertebrate Hbs, possibly reflecting the various demands to be met among their hosts. Sharing structural features with other haem proteins/enzymes, Hbs display a qualitative overlap in activities with functions extending beyond oxygen transport/storage.



When subjected to oxygen deficient conditions, the obligate aerobic bacterium Vitreoscilla synthesises a homodimeric haemoglobin, VHb, envisaged to sustain respiratory growth by facilitating oxygen-delivery to the respiratory apparatus. Heterologously expressed in various host organisms, VHb has shown to exert positive effects on growth and product formation. Furthermore, results from recent studies imply that VHb may be involved in alleviating oxidative/nitrosative stress. Despite being extensively studied, the biological function(s) of VHb remains elusive.



This thesis describes two approaches aiming to gain further knowledge about the potential functions and biotechnological usefulness of VHb, including both its ability to exhibit peroxidase activity in vitro and the effect of VHb expression on growth and metabolism in aerated cultures of Lactococcus lactis and salt-stressed cultures of Escherichia coli. Our investigation reveals for the first time that VHb does exhibit peroxidase activity comparable to other peroxidases. In addition, studies of mutant VHbs and an engineered single-chain dimer, double VHb (dVHb), show that the catalytic activity can be modified by local alterations in the proximal site structure without perturbation of the globin fold. This property may be exploited in various biotechnological applications; the preferred VHb variant could be chosen depending on the conditions of interest.



The in vivo studies of VHb in L. lactis describe the hitherto uninvestigated expression of a haemoglobin in a host organism lacking fully functioning respiration capacity. Our results show that in aerated cultures supplemented with haemin, L. lactis cells expressing biologically active VHb reach higher final cell densities than control cells. Expression of native VHb and an engineered single-chain dimer, double VHb (dVHb), in E. coli subjected to high concentrations of NaCl (up to 1.1 M) result in higher final cell densities for dVHb-cells than control cells lacking VHb for all conditions studied. Furthermore, our results imply that dVHb-cells show less signs of oxidative stress than control cells. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Wilson, Michael, Dept. of Biological Sciences, University of Essex, United Kingdom
organization
publishing date
type
Thesis
publication status
published
subject
keywords
metabolism, Biokemi, Metabolism, Biochemistry, Naturvetenskap, Natural science, lipid peroxidation, hypoxia, salt stress, Escherichia coli, Lactococcus lactis, hydrogen-bonding network, proximal site, mutations, Vitreoscilla haemoglobin, peroxidase activity, Proteins, enzymology, Proteiner, enzymologi
pages
148 pages
publisher
Tillämpad biokemi, Lunds Universitet
defense location
Kemicentrum Sölvegatan 30 Lund
defense date
2007-02-01 10:15
ISBN
978-91-7422-137-4
language
English
LU publication?
yes
id
ba2fceb0-b6cd-4d1f-bfb7-a8656c03950f (old id 547866)
date added to LUP
2007-10-09 11:49:14
date last changed
2016-09-19 08:45:13
@misc{ba2fceb0-b6cd-4d1f-bfb7-a8656c03950f,
  abstract     = {The function of non-vertebrate haemoglobins (Hbs) appears more diverse than that of vertebrate Hbs, possibly reflecting the various demands to be met among their hosts. Sharing structural features with other haem proteins/enzymes, Hbs display a qualitative overlap in activities with functions extending beyond oxygen transport/storage.<br/><br>
<br/><br>
When subjected to oxygen deficient conditions, the obligate aerobic bacterium Vitreoscilla synthesises a homodimeric haemoglobin, VHb, envisaged to sustain respiratory growth by facilitating oxygen-delivery to the respiratory apparatus. Heterologously expressed in various host organisms, VHb has shown to exert positive effects on growth and product formation. Furthermore, results from recent studies imply that VHb may be involved in alleviating oxidative/nitrosative stress. Despite being extensively studied, the biological function(s) of VHb remains elusive.<br/><br>
<br/><br>
This thesis describes two approaches aiming to gain further knowledge about the potential functions and biotechnological usefulness of VHb, including both its ability to exhibit peroxidase activity in vitro and the effect of VHb expression on growth and metabolism in aerated cultures of Lactococcus lactis and salt-stressed cultures of Escherichia coli. Our investigation reveals for the first time that VHb does exhibit peroxidase activity comparable to other peroxidases. In addition, studies of mutant VHbs and an engineered single-chain dimer, double VHb (dVHb), show that the catalytic activity can be modified by local alterations in the proximal site structure without perturbation of the globin fold. This property may be exploited in various biotechnological applications; the preferred VHb variant could be chosen depending on the conditions of interest.<br/><br>
<br/><br>
The in vivo studies of VHb in L. lactis describe the hitherto uninvestigated expression of a haemoglobin in a host organism lacking fully functioning respiration capacity. Our results show that in aerated cultures supplemented with haemin, L. lactis cells expressing biologically active VHb reach higher final cell densities than control cells. Expression of native VHb and an engineered single-chain dimer, double VHb (dVHb), in E. coli subjected to high concentrations of NaCl (up to 1.1 M) result in higher final cell densities for dVHb-cells than control cells lacking VHb for all conditions studied. Furthermore, our results imply that dVHb-cells show less signs of oxidative stress than control cells.},
  author       = {Kvist, Malin},
  isbn         = {978-91-7422-137-4},
  keyword      = {metabolism,Biokemi,Metabolism,Biochemistry,Naturvetenskap,Natural science,lipid peroxidation,hypoxia,salt stress,Escherichia coli,Lactococcus lactis,hydrogen-bonding network,proximal site,mutations,Vitreoscilla haemoglobin,peroxidase activity,Proteins,enzymology,Proteiner,enzymologi},
  language     = {eng},
  pages        = {148},
  publisher    = {ARRAY(0x8093228)},
  title        = {Vitreoscilla Haemoglobin - Peroxidase Activity and Metabolic Aspects},
  year         = {2007},
}