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


CLASSIFICATION OF RUBISCOS

Form I - Carbon metabolism Form II – Carbon metabolismForm III – AMP metabolism Form IV

Methionine Salvage PathwayEvolutionary progenitor

RuBP dependent


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


Table–Classification of Rubiscos


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)


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.


FORM I RUBISCOs (ctd.,)

distinguishing feature of the Form IAq gene arrangement - universal presence of two genes downstream of the LS gene pair


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


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.


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.


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

structural analysis of co2 binding motif

Science