Functional significance of an overlapping consensus binding motif ...
Structural analysis of CO2 binding motif
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Transcript of Structural analysis of CO2 binding motif
STRUCTURAL ANALYSIS OF CO2 BINDING MOTIF
Presented by Vamanie K P
(A model for the structure of rubisco in chloroplasts from higher plants. Rubisco consists of 8 large (L) and 8 small (S) subunits arranged as 4 dimers. Small subunits are shown in red (only four of the small subunits are seen), large subunits are shown in blue and green, in order to show the boundaries of the dimers. (From Malkin and Niyogi 2000.)
Preliminary step to improve the photosynthetic efficiency of Rubisco.In silico comparative analysis of sequence and structure for the prediction of residues affecting CO2 binding affinity of Rubisco. In silico comparative analysis of CO2 and O2 binding domains of other enzymes with that of Rubisco.Review on Rubisco and CO2 binding domains
OBJECTIVE
Carbon FixationCO2 Fixation Pathways
Calvin Benson Bassham pathway Reductive citric acid cycle Reductive acetyl Co A pathway 3 – hydroxypropionate bicycle hydroxypropionate-hydroxybutyrate cycle dicarboxylate-hydroxybutyrate cycle
The incompetent Rubisco enzyme Classification of RubiscosApproaches to improve photosynthetic efficiency of Rubisco in practice
INTRODUCTION
CO2 FIXATION PATHWAYS Carbon – inorganic Form Fixed by
autotrophicorganisms
Tabita et al., 2007
CBB PATHWAY
aerobic autotrophic bacteria Autotrophic plants
Menendez et al., 1999
RUBISCO
GENERAL INFORMATION ABOUT THE PROTEIN AND CLASSIFICATION
RUBISCO 1, 5 Ribulose bisphosphate
Carboxylase/ Oxygenase. RubisCO - large (catalytic) and small
subunits to form a massive hexadecameric protein structure with an Mr of about 550,000, i.e., eight copies of both large ( 55,000 Mr) and small ( 15,000 Mr) polypeptides in an (L)8(S)8 structure.
Tabita et al., 2007
The genes coding for LSU are usually found in the chloroplast and those genes for SSU are nuclear.
The SSUs are transported from the nucleus to the stroma of the chloroplast during assembly of the sub units, thus forming the entire protein.
Mg2+ ions are essential for the enzyme activity- appendage of an activated CO2 molecule to the lysine residue at the active site and culminating in the formation of carbamate
(Chatterjee et al.,) large sub - unit contains the α/β barrel active site small sub - unit genes influence the carboxylation
rate and the CO2 / O2 specificity (Genkov et al., 2010).
THE INCOMPETENT RUBISCO ENZYME
Catalytic turn over rate in terms of CO2 fixation
Low affinity for CO2 O2 – a competitive substrate
Badger and Bek, 2008
EVOLUTION OF RUBISCOs
Changes in Km and CCMs Major driving force
Changes in the concentration of CO2 and O2
O2 concentration 35% , now 21% CO2 concurrent decline from >1% to
0.02% 280 Mya and now on the rise 0.037%
Nitrogen scarcity
Badger and Bek, 2008
CLASSIFICATION OF RUBISCOS
Form I - Carbon metabolism Form II – Carbon metabolismForm III – AMP metabolism Form IV
Methionine Salvage PathwayEvolutionary progenitor
RuBP dependent
Badger and Bek, 2008
Well recognis
ed function
Doubted
FORM I
Green I A I Ac
I Aq
I BI B
I Bc
RedI C
I D
FORM IIFORM III
FORM IV
Badger and Bek, 2008
Table–Classification of Rubiscos
Badger and Bek, 2008
FORM I RUBISCO Forms IA and B ‘Green’ found in
proteobacteria, cyanobacteria, green algae, and higher plants.
Forms IC and D ‘Red’ proteobacteria and non-green algae.
Form IA enzymes two distinct types IAc and IAq, based on two distinct types of small subunits and gene arrangements.
Form IB enzymes subclassified into IB and IBc (cyanobacteria which is associated with Carboxysomes)
Badger and Bek, 2008
FORM I RUBISCOs (ctd.,) IAc – associated with carboxysomes IAq – associated with cbbQ1 and cbbO1
gene Major difference between the proteins – is
in the genes coding for small subunits of the L8S8 enzymes (observed in bacterial proteins and genes)
All Form IAq small subunit sequences known till date are characterized by a 6 aa insertion at the N-terminal end of the protein.
Badger and Bek, 2008
FORM I RUBISCOs (ctd.,)
distinguishing feature of the Form IAq gene arrangement - universal presence of two genes downstream of the LS gene pair
Badger and Bek, 2008
FORM I RUBISCOs (ctd.,)
Rubisco Form Ic LS genes are found in association with cbbX located downstream.
codes for a protein of unknown function
Is thought to be involved with some form of posttranslational activation of the Rubisco
Badger and Bek, 2008
FORM II RUBISCO
Rubisco II was originally isolated as a second peak of activity after ion-exchange fractionation of extracts from induced R. sphaeroides.
Form I RubisCO was isolated from the same crude extracts, i.e., in the first activity peak that eluted from the column.
Thus came the names I and IITabita et al., 2007
Ctd., form II enzyme - multimers of large-
type subunits [(L2)x], shows only about 30% amino acid sequence identity to form I large subunits.
less efficient in partitioning the two gaseous substrates
Tabita et al., 2007
FORM III RUBISCO Archael Rubiscos the lack of any demonstrable
phosphoribulokinase (PRK) activity (or a gene that encodes this protein) from these same organisms that contain RubisCO has been a major curiosity
Provides insights into the structure-function relationships
Obtained from organisms that never see molecular oxygen
offered tantalizing possibilities to learn more about how the active site of RubisCO might have evolved.
Tabita et al., 2007
FORM IV RUBISCO they fail to cluster with any bona fide
RubisCO sequence in phylogenetic trees and that each sequence contains nonconservative substitutions at the positions normally occupied by conserved RubisCO active-site residues
about 35% identity at the amino acid level. participates in a methionine salvage pathway
and catalyzes the enolization of the RuBP Analog 2,3-diketo-5-methylthiopentyl-1-P.
Six clades.Tabita et al., 2007
Ctd., The six clades in the RubisCO form IV (RLP)
lineage termed as IV-Photo (found in phototrophic bacteria), IV-NonPhoto (found in nonphototrophic bacteria), IV-AMC (acid mine consortia), IV-YkrW, IV-DeepYkr, IV-GOS (global ocean sequencing sequencing
program) based on characteristics of the source organisms,
prior designation of the gene product, and/or relationship to other sequences
Tabita et al., 2007
Mutations in RLPs result in deficient sulfur oxidation and increase in production of stress responsive elements.
Tabita et al., 2007
detailed functional studies have been carried out for only four RLPs, C. tepidum RLP, the YkrW/MtnW proteins of Bacillus subtilis and Geobacillus kaustophilus, and the YkrW-like RLP from the cyanobacterium Microcystis aeruginosa.
the three-dimensional structures have been solved only for C. tepidum RLP , R. palustris RLP2 , and G. kaustophilus RLP
Tabita et al., 2007
The RLP from C. tepidum and the RLP2 from R. palustris are structurally very similar at the active site but possess four different active-site residues compared to the B. subtilis proteins.
Specific catalytic residues differentially conserved among the two lineages.
The major difference - Glu versus the Lys at Asn-123 (spinach RubisCO numbering), suggesting possible differences in hydrogen-bonding patterns with their respective substrates.
In addition, Asn versus Val/Met identities at the Lys-177 position in C. tepidum versus B. subtilis groups of RLPs, respectively, may indicate different needs or participants for proton abstraction at the presumptive active site
whereas Phe versus Pro identities at Arg-295, the residue that interacts with P2 phosphate in spinach RubisCO, likely indicate that each type of RLP reacts with distinct substrates with different hydrophobicities at the P2 site.
Tabita et al., 2007
CONSERVED RESIDUES AND MOTIFS IN RUBISCOs four residues absolutely conserved among all
members of the RubisCO superfamily Gly-122/110/100, Lys-175/166/153, Asp-
203/193/191, and Gly-322/316/297 in representative enzymes of form I, form II, and form III from Spinacia oleracea, Rhodospirillum rubrum, and Methanocaldococcus jannaschii, respectively.
99% conservation - identification of 10 additional residues.
Three of these highly conserved residues, Asp-198/188/176, Lys-201/191/179, and Asp-203/193/181, lie within the “RubisCO motif.” Tabita et al.,
2007
Lys-201/191/179 is the residue that becomes carbamylated when RubisCO is “activated” by CO2 in the presence of a divalent metal when the RubisCO-CO2-Me2 ternary complex is attacked by a second molecule of CO2 or O2.
Lys-175/166/153 is involved in the initial deprotonation and final protonation steps of the catalytic cycle.
90 % identity in all Rubiscos and RLPs -0 Asp-203/193/181 is one of the key metal binding ligands along with Glu-204/194/ 182,
highly conserved glycines have not been ascribed specific roles in RubisCO structure or function.
Tabita et al., 2007
25 residues at 90% cut off for conservation account for the conserved functions among all RubisCO large-subunit sequences are Mg2 binding, acid-base chemistry, substrate hydration, and a partial P1 binding site.
18 conserved but non-active-site residues may well reflect unrecognized players in catalysis or protein stability or keystone residues critical to establishing or maintaining the structure of the active enzyme.
Tabita et al., 2007
“ The overall monomer structures of all RubisCO large-subunit superfamily members are quite similar and suggests that there may be a conserved set of residues that are critical for folding and maintaining this general structure.
the authentic RubisCO proteins (forms I, II, and III) all catalyze the same reactions, may have widely different enzymatic properties
even proteins whose structures are virtually superimposable, with up to nearly 90% sequence identity, may possess vastly different kinetic properties.
Tabita et al., 2007
active site of RLP is located in the subunit interface between the C-terminal domain of one subunit and the N-terminal domain of another subunit
Loop 6 in the C-terminal /-barrel domain of RubisCO - plays an important role in catalysis
Tabita et al., 2007
APPROACHES TO IMPROVE PHOTOSYNTHETIC EFFICIENCY OF RUBISCO IN PRACTICE
Manipulation of Rubisco activation by increasing levels of Rubisco activase.
Enhancing catalytic efficiency and specificity of Rubisco.
Increasing the regenerative capacity of the RuBP in the CBB pathway.
Introduction of Carbon Concentrating Mechanisms into higher plants.
Redistribution of enzymes in the CO2 fixation pathway. Design and Introduction of a new synthetic CO2
fixation pathway in plants. Understanding mechanisms underlying natural
variations in Rubiscos in response to different environments
(Raines, 2011).
Selection of Rubiscos with varying kinetic properties Comparative analysis of Rubisco genes Comparative analysis of 1º, 2º and 3º structures of Rubiscos Selection of enzymes for which CO2 and O2 are substrates Comparative analysis of CO2 and O2 binding domains with Rubisco
METHODOLOGY
RUBISCOS WITH DIFFERENT KINETIC PROPERTIES Kinetic properties depend on the
exact nature of the CO2 environment in which Rubisco operates
When external CO2 high during the CO2-fixation period or there is intervention of a CCM, then it is likely that higher Km values for CO2 fixation will have evolved.
Badger and Bek, 2008
Ctd., Form II Rubiscos - low Selectivity value, high
Km for CO2, relatively high kcat. They have evolved in low-O2 and high-CO2
environments. Form Ic enzymes show improved Selectivity
values and medium to low affinities for CO2, indicates an adaptation to environments with
medium to high CO2 but with O2 present. Form IBc Rubiscos well studied have medium
Selectivity values and the lowest affinities for CO2. a well-characterized b-carboxysome-associated CCM.
Badger and Bek, 2008
Ctd., Form ID enzymes show high
Selectivity and low Km values for CO2. Form IAq enzymes adapted to medium
to high CO2 but with O2 present. Form IAc Rubiscos poorly studied
kinetic properties. have Selectivity, values that are less
than that for Form IBc, medium to low affinity for CO2.
this is based on a very limited sampleBadger and Bek, 2008
SCOPE OF THE PROJECToverall efficiency of photosynthesis very low
the catalytic step of Rubisco is very slow
increased production of biomass
economic outgrowth and solutions for dearth in food availability.
“FURTHER IN VITRO AND IN VIVO STUDIES
WOULD COME UP BASED ON THIS COMPUTATIONAL ANALYSES
AND ARE REQUIRED TO VALIDATE THE OBSERVATIONS OBTAINED IN
THIS STUDY”
COLLECTION OF ENZYMES
Search for enzymes with the following information in order
Kinetic parameters – especially Km values for CO2 and O2, selectivity
Sequence information for the gene, protein
Structure of the protein
Kinetic parameters information collected from…BRENDA database – for enzymes
(Schomburg et al., 2002)Research article – Badger and Bek
2008
# of Rubiscos for which information was available = 50
Collection of sequence and structure information for the Rubiscos Gene , protein and structure
information was searched in NCBI and EMBL
accordingly Rubiscos from the following organisms were selected
Structure available only for those marked in pink
1. Coffea Arabica2. Oryza sativa3. Nicotiana tabacum4. Griffithsia monilis5. Triticum aestivum6. Spinacia oleracea7. Hydrogenophaga
pseudoflava8. Porphridium purpureum9. Chlamydomonas
reinhardtii10.Allochromatium vinosum
1. Thermosynechococcus elongatus
2. Cupriavidus necator3. Synechococcus PCC70024. Bradyrhizobium
japonicum5. Rhodospirillum rubrum6. Thiobacillus
denitrificans 7. Rhodobacter
sphaeroides8. Rhodobacter capsulatus9. Xanthobacter flavus
BLAST done for
Gene sequence Protein sequence
And shaded alignment file obtained from BoxShade server
Analysis of the BLAST resultsStructure alignment and analysisReview on CO2 binding domainsComparison with other enzymes using CO2 as substrate.
WORK TO BE DONE