MAIN
©1996-2006 All Rights Reserved. Online Journal of Bioinformatics . You may not store these pages in any form except for your own personal
use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way
besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJB
publications. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors.
This journal satisfies the refereeing requirements (DEST) for the Higher Education Research Data Collection (Australia). Linking:To link to this
page or any pages linking to this page you must link directly to this page only here rather than put up your own page.
OJB
Online Journal of Bioinformatics ©
TM
7 (1): 35-45, 2006
Comparative structure analysis of Chorismate synthase
Marla S , Yalamanchili HK, Gelli P, Singh HK, Praveen G,
Ghatta G, Srikanth S, Goutham K.
Biotechnology and Bioinformatics Department, JayaPrakash University of Information technology, Solan, HP, India.
ABSTRACT
Marla S , Yalamanchili HK, Gelli P, Singh HK, Praveen G, Ghatta G, Srikanth S, Goutham
K Comparative structure analysis of Chorismate synthase, Online Journal of
Bioinformatics, 7 (1) : 35 - 45, 2006.This work compares chorismate synthase structure
in various microorganisms. The enzyme is essential in the shikomate pathway in
bacteria, fungi and plants, but not in mammals. It is a useful target for drug design
inhibiting functional pathways with chemotherapeutic effects. Binding of essential cofactors Flavin mononucleotide (FMN) and 5-enolpyruvil shikomate-3-phosphate(EPSP)
affects its activity. The results of FMN structure analysis and in various microorganisms
are presented. Comparative modeling and extent of structure similarities between H.
pyroli and P. rumnicola were performed using alpha and beta fold patterns, FMN
binding and main chain configuration residues. Active sites for binding of FMN were
detected. Phylogeny analysis was evaluated in various microorganisms.
Key words: Chorismate synthase, flavin mononucleotide, Binding sites, Helicobacter
pyroli, Prevotella rumnicola.
35
INTRODUCTION
Helicobacter pyroli is responsible for causing several water born diseases in humans
including type-B gastritis, peptic-ulcer and lymphoid-tissue (MALToma: Alm, R.A., et al.,
2001: Blaser 1992: Wotherspoon et al., 1993). The appearance of antibiotic-resistant H.
pyroli strains poses a threat to disease therapeutics. In this work, the identification and
characterization of molecular targets associated with H .pyroli and P. rumnicola, a
pathogenic bacterium responsible for gastritis and MALToma is presented.
H. pyroli is involved in the pathogenesis of gastric mucosa associated with lymphoidtissue (MALToma) (Wotherspoon et al. 1993). The long chain of flavodoxin of H. pyroli
functions as an electron acceptor to the Peruvate-oxidoreductase (POR) enzyme in
chorismate metabolism (Hughes et al. 1995; Kaihovaara et al. 1998). It was also
discovered that sera from patients with gastric MALToma contained antibodies against
a 19 kD protein flavodoxin (Chang et al. 1999; Shiesh et al. 2000). H. pyroli encodes
flavodoxins- acidic redox proteins belonging to flavin mononucleotide (FMN) containing
proteins involved in a variety of electron transfer reactions (Tomb et al. 1997: Alm et al.
1999).
Chorismate synthase enzyme from the shikomate pathway has an absolute
requirement for reduced flavin mononucleotide for the synthesis of aromatic amino
acids, though the former is not consumed in the reaction (Kitzing et al.2004).
Chorismate is used as a substrate for other pathways that lead to synthesis of folates,
ubiquinones, napthoquinones and amino acids like phenylalanine, tryptophan and
tyrosine. The shikomate pathway and flavodoxins are present in both eukaryotes and
prokaryotes but not in mammals (Osborne et al. 1991; Romero et al. 1996).
The shikomate pathway is also present in apicomplexan parasites like taxoplasma
gondii, plasmodium falciforum, cryptosporidium parvum, Clostridium difficile (Roberts et
al. 2002: Sunita et al. 2004). The discovery of potential inhibitors of the shikomate
pathway may help to design a spectrum of antimicrobial agents that are effective
against bacterial, fungal pathogens and apicomplexan parasites. The fldA gene in
genome of H. pylori encodes the small acidic redox protein flavodoxins (Tomb et al.
1997: Alm et al., 1999). The flavodoxins are flavin mononucleotide (FMN) containing
proteins that are involved in a variety of electron transfer reactions.
The shikomate pathway is essential for synthesis of many compounds starting from
carbohydrates to biosynthesis of aromatic compounds such as Tryptophan, Tyrosine,
vitamins K, P and amino benzoic acid (PABA). PABA is later converted in the reaction to
folates. The pathway uses phosphoenol pyruvate and erythrose in seven catalytic steps
to synthesize chorismate (Hermann et al., 1999, Knaggs, 2001). Whereas in parasitic
bacteria the shikomate pathway is essential for their survival (Roberts et al., 2002 ).
36
Seven enzymes of the Chorismate pathway are involved in sequential conversion of
erythrose 4-phosphate and phosphoenol pyruvate to chorismate. Chorismate synthase
catalyzes this reaction by eliminating the 3-phosphate group and the C-(6proR)
hydrogen from 5-enolpyruvylshikamate 3-phosphate (ESPS) to yield chorismate.
Although this reaction does not involve a net redox change, the enzyme has an absolute
requirement for reduction of flavin mononucleotide (FMN). However Flavin
mononucleotide is not consumed in the reaction (Kitzing et al., 2004). FMN (Flavin
Mononucleotide) C17H21N4O9P is a derivative of riboflavin that condenses with adenine
nucleotide to form flavin adenine dinucleotide and acts as a coenzyme for various
flavoproteins in oxidation-reduction reactions in cells. Flavodoxins can be divided into
two structural classes: short and long chain flavodoxins (Mayhew and Ludwig 1975).
Long-chain flavodoxin of H. pylori acts as an electron acceptor to the POR enzyme
complex, which catalyzes the oxidative decarboxylation of pyruvate (Kaihovaara et al.
1998).
Genes encoding various enzymes in shikomate pathway including chorismate synthase
in fungi and plants have already been reported , but in microorganism to date a few are
only reported (Davies et al, 1994: Roberts et al, 1998: Sunita et al., 2004). Recently,
evidence for the presence of chorismate synthesis in Thermodesulfobacteriales (gi:
70907682), Holoferax volcani (gi: 68146591), Dichelobacter nodosus (gi:68146596),
Thrmodesulfobacterium commune.(gi: 70907682 ) and Salinibacter rubber (gi:
68146594) has been found.
P. rumnicola is a gram-positive obligate anaerobic bacterium chiefly inhibiting in the
rumen of live stock is also involved in the infection of bowel and gastroentesis in
humans (Blaser, 1992: Nicolich et al., 1992). The genome of P. rumnicola is not yet been
fully sequenced (TIGR, 2005) but is being annotated. The bioinformatics of this enzyme
provides tools to develop drugs as potential antibacterial drugs.
We present here our work on comparative modeling of chorismate synthase from
Prevotella rumnicola based on the crystal structure of chorismate synthase from H.
pyroli.
RESULTS and DISCUSSION
The enzyme structure was deduced from multiple sequence analysis and location of
conserved residues and active sites. The detected active binding sites of the enzyme
were further validated by a Ramachandran plot. To deduce the evolutionary
relationships among the above described genomes we conducted phylogeny analysis.
Multiple sequence analysis was conducted to discover the existing conserved patterns
of chorismate enzyme in various microorganisms. PROSITE database analysis (Falquet et
al. 2002) revealed high order conservation of Casein Kinase II Phosphorylation site
(CK2_PHOSPHO_SITE), N-myristaolation site (MYRISTYL), Protein Kianase C
phosphorilation site (PKC_PHOSPHO_SITE), Tyrosine Sulphation (SULPFATION) and N37
glycosylation (ASN_GLYCOSYLATION) sites in H. pyroli, P. rumnicola, H. volcani, and S.
rubber genomes. How ever D.nodosus and T. commune also contained Nmyristaolation, and N-glycosylation but did not contain Casein Kinase II Phosphorylation,
Protein Kianase C phosphorilation and Sulphation sites. Unlike H. pyroli , P.rumnicola
did not contain Tyrosine sulphation sites.
Pair wise sequence analysis of H. pyroli and P. rumnicola sequences was done using
DOTPLOT (Maijel et l, 1981). Analysis data revealed an existence of similarity of 50.0
percent and an identity of 36.34 percent. BLAST search was done against SWISS-PROT
database using the chorismate synthase amino acid sequence from H. pyrol. BLAST
search revealed existence of more than 60 homologous chorismate equences from
various organisms. ClustalW output showed three patterns conserved in all the studied
organisms. Two binding sites -histidine residues, His 106 and His 17 were found in the
active site of the enzyme flavin mononucleotide.
Structural information: The crystal structure of chorismate synthase from Helicobacter
pyroli was determined recently at 1.95A0 resolution( PDB ID: 1UMO: Freigang, J. et al.,
2002). This information was used as a template for elucidating the structure of P.
rumnicola.
The predicted homology model of chorismate synthase from P. rumnicola showed a
tetrameric enzyme structure with each monomer possessing a novel three layered
“beta-alpha-beta sandwich fold”. The observed folds were found to be highly conserved
in H. pyroli. We compared the observed FMN structure with that of the crystal
structure of H. pyroli. In our model all the FMN binding residues were found to located
in the similar loop regions (F1 –F6 regions) similar to the crystal structure of H. pyroli.
The electrostatic potential around the binding sites of FMN was observed to be highly
positive. This was confirmed in our study that the FMN structure of P. rumniola exist in
similar positions as shown in the crystal structure of H. pyroli i.e. in all six flexible loop
regions starting from F1 to F6. Incidentally these regions were observed to be rich in
highly conserved residues like Argenine. Presence of highly conserved residues confirm
the fact that show that active site are almost similar in both H. pyroli and P. rumnicola
(Table.1).
Table. 1. Residues at FMN binding site (Ligplot analysis results).
Positions in 1UMO , in H. pyroli
Arg  123
Arg  330
Ser 125
Lys  296
Positions in P. rumnicola
Arg  121
Arg  333
Ser 125
Lys  296
38
Figure 1. Location of four active site binding FMN in chorismate synthase in P.
rumnicola (MOE V.11.0).
The obtained results further confirm the presence of close similarities in FMN binding in
P. Rumnicola and H. pyroli. Protein structure was further probed inferred
usingpackages Molecular Operating Environment(MOE v. 11.0) and Modeler (Insight II).
model generated by Modeler was based on sequence alignment between H. pyroli and
P. rumnicola. Modeler employed a probability density functions as the spatial restraints
( Sali et al. 1995) to compare to the energy functions generated by MOE.
The main-chain confirmation of a residue location was found by noting the restraints
which depend on residue type and main chain matching amino acid residues in the
reference proteins. Protein databases information (PDF) of homologous protein
families based on the structure information was used to define the probability density
functions in restraining the model structure. The PDF are employed to restrain C – C
distances, main chain N-O distances, main chain and side chain dihedral angels. The
individual PDF assembled in to a have its semantics in terms of energy functions in a
molecular mechanics force field functions. PDF along with the coordinate information
is retrieved from Protein Data Bank(PDB) from H. pyroli (1UDB) was used to generate
the molecular probability functions. An optimum 3D structure of P.rumnicola was
arrived using the available molecular probability functions. The structure of oxidized P.
rumnicola flavodoxin was solved by molecular replacement to 2.4 Å resolution. The
protein has an /ß-fold, and also shares a high degree of similarity with the other
structurally known flavodoxins, from H. pyroli (Fig. 4). Similarly a total of five helices (
1– 5) were observed (Fig. 2) and it was noted that ß strands (ß1–ß5) form a central ßsheet and Helices 1 and 5 are on one side of the ß-sheet, and helices 2, 3, and 4
are located on the opposing side (Fig. 2).
39
Strand ß5 is interrupted by an insertion of 18 residues, that make the P. rumnicola
protein a long-chain flavodoxin similar to that of H. pylori. The binding site for the
cofactor FMN is located at the carboxy-terminal end of the ß-sheet. Superposition of
these structures revealed (Fig.4) that P. rumnicola flavodoxin closely resembles the
protein from H. pylori. However the C -trace of the P.rumnicola protein differs in two
loop regions significantly from the C -traces of the protein H. pylori. The 2–ß3 loop
contains one residue less than the H. pylori. Similarly, the 4–ß5 loop contains two
residues less than the equivalent loops of the other flavodoxins (data not shown). The
observed difference in architecture may affect the neighboring loops, which in turn
adopt a conformation significantly different from the one found in H. pyroli. However,
the above listed differences in the and ß loops are unlikely to affect the active site, as
the distance from the loops at the amino-terminal end of the central ß-sheet to the
cofactor is larger than 15 Å.
Figure 2. Secondary structure of Chorismate synthase in P. ruminicola.(From Insight II,
Secondary render).
Figure 3. Quaternary Structure of Chorismate synthase in P. ruminicola.(From Insight II)
40
Figure 4 A & B.Backbone of P. rumnicola and Structural Superimposition of Chorismate
synthase of H .pyroli Vs P.ruminicola. (Green-H. pyroli and red-P. rumnicola, MOE. V.
11.0)
Evaluation of the protein structure by Procheck:The Predicted model of P. rumnicola
chorismate synthase was verified using Procheck, a program employing stereochemical
properties of proteins.
A Ramachandran plot showing phi-psi angles is obtained (Figure 5) .The percent residues
in the favorable region [A,B,L] is 79.2 (Figure 3) and 18.2 % in the additional allowed
regions, which is comparable to that of crystal structure of H. pyroli (click to enlarge
figures 4-).
Figure 5. Evaluation of protein structure. Procheck illustration of Ramachandran Plot.
41
Phylogenic analysis:Chorismate synthase is mono functional as it does not possess the
ability to reduce FMN cofactor and thereby reflects the enzyme’s ancestral origin. To
study the evolutionary relationship of chorismate synthase gene we compared the
chorismate synthase genes in H. pyroli and P. rumnicola with other chorismate synthase
genes we identified in organisms viz. Helicobacter volcani, Salinibacter ruber,
Thermodesulfobacterium commune and Dicheloro nodosus. Prosite analysis revealed
that Salinibacter rubber lacked Casein kinase II phosphorilation, C-phosphorylation sites
compared to H. pyroli. This fact reflects the divergence observed in Phylogenic analysis
(Fig. 6).
It was reported that the Phosphate group is bound by a loop that contains the key
fingerprint motif, for flavodoxin, T/S-X-T-G-X-T (Drennan, 1999). In future we are
planning to conduct studies to identify fingerprint motifs unique to chorismate
synthase o each micro organism analyzed.
Fig. 6. Tree View Tool in CLUSTALW
Gene identification from H. rumnicola genome: The genome of H. rumnicola strain 23 is
a gram-negative, anaerobic gastrointestinal bacterium and the genome is being
sequenced and Random shotgun sequencing is underway (TIGR, 2005),. It is a non
pigmented, strongly fermentative species isolated from the rumens of cattle, sheep, and
elk, and from human abscesses and feces.
It is one of the pathogenic bacteria that cause a self-limiting gastroenteritis in humans
(Nikolich et al. 1994). Chorismate synthase amino acid sequence of Helicobacter pyroli
was retrieved from Swissprot databank and compared with the genome of P. rumnicola
using TBLASTN. Chorismate synthase gene was found to be reverse complimentary
fashion and was reverse complimented using on-line tool REVERSE TRANSLATE. We have
also identified chorismate synthase genes from other micro organisms and submitted
with EMBL database as described above.
42
CONCLUSIONS
Chorismate synthase in microorganisms was found to have high levels of sequence as
well as structural homology similar to Helicobacter pyroli. The predicted homology
model of chorismate synthase from P. rumnicola showed a three layered “beta-alphabeta sandwich fold” with highly conserved regions that include several flexible loops
clustered together to form an active site with a unique FMN binding pocket. The loop
regions of FMN binding residues were found to be similar to the crystal structure of H.
pylori with minor variations. The observed homology model was verified using
PROCHECK and was 79.2% accurate to the predicted model. Knowledge of the
architecture of FMN binding sites in P. rumnicola improves our understanding of the
actions of inhibitors of the shikomate pathway delivered to the chorismate synthase
enzyme.
Acknowledgments:
We thank Ms. Sunita, GVK BIO, Hyderabad, India for providing training facility, and also
grateful to JUIT for providing required facilities and support.
REFERENCES
Ahn H.J., Yoon H.J., Lee B, suh sub.2004. Crystal structure of chorismate synthase. a novel FMNbinding protein fold and functional insights . J. Mol.Biol.. 27;36(4):903-15.
Alm.R.A. ling, L.S. Mair, D.T. etc.1999.
Genomic-sequence comparisons of two unrelated isolates of the human gastric pathogen
Helicobacter pyroli.nature 397:176-80.
Blasev,M.J.1992.hypothesis on the pathogenesis and natural history of Helicobacter pyroliinduced inflammation .Gastroentology .102:720-7.
Brooks, B.R.,Bruccdevi,R.E.,1993 Olafson,B.D.,States,
D.J.,Swaminathan,S.,Karpulus,M.CHARMM:A program for macromolecular energy,
minimization, and dynamic calculations. J.compat Chem.4,187-195.
Chang, C.S., Chen, L.T., Yang, J.C., Lin, J.T., Chang, K.C., Wang, J.T., 1999 Isolation of a
Helicobacter pylori protein, FldA, associated with mucosa-associated lymphoid tissue
lymphoma of the stomach. Gastroenterology. 117:82-8.
Davies, G.M., Barret-Bee, K.J., Jude, J.A., et al, 1994. (6S)- 6 Fluroshikomic acid, an antibacterial
agent acting in the aromatic biosynthetic pathway. Antimicrobe Agent Chemother. 38: 403.
Drennan, C.L., Pattridge, K.A., Weber, C. H., Metzger, A.L., Hoover, D.M. . and Ludwig, M.L.
1999.Refined structures of oxidized flavodoxin from Anacystis nidulans. J. Mol. Biol. 294:
711-24.
43
Falquet, L., Pagni, M., Bucher, P., Hulo, N., Sigrist, C.J.A., Hofman, K. and Bairoch, A.. (2002). The
PROSITE database , Nucleic Acids Res., 30, 235-23
Freigang, J., Diederichs ,K., Schafer, K.P., Welte, W., Paul, R. 2002 Crystal structure of oxidized
flavodoxin, an essential protein in Helicobacter pylori. Protein Science.11:253-261.
Hermann, L., Schwan, D., Garner, R., Mobley, H.L., Haas, R., Schafer, K., Melchers, K., 1999.
Helicobacter pylori cadA encodes an essential Cd(II)-Zn(II)-Co(II) resistance factor influencing
urease activity.Mol Microbiol.33:524-36.
Hughes, N.J., Clayton, C.L., Chalk, P.A., Kelly, D.J. 1998. Helicobacter pylori por CDAB and
oorDABC genes encode distinct pyruvate: flavodoxin and 2-oxoglutarate:acceptor
oxidoreductases which mediate electron transport to NADP.
J Bacteriol. Mar;180:1119-28.
Insight II,version 2000.2001. SanDiego:Accelarys inc.
(http://www.accelarys.com).
Kaihovaara, P., Hook-Nickname, J., Uusi-Oukari, M., Kosunen, T.U., Salaspuro, M.1998,
Flavodoxin-dependent pyruvate oxidation, acetate production and metronidazole reduction
by Helicobacter pylori. J. Antimicrob. Chemother.41:171-7.
Kitzing, K., Auweter, S., Amrhein, N., Macheroux, P. Mechanism of chorismate synthase. Role of
the two invariant histidine residues in the active site.
J Biol Chem. 2004 5;279(10):9451-61.
Knaggs, A.R.,2001 The biosynthesis of shikomate metabalites. Nature. Prod. Rep. ; 18: 334-355.
Maizel JV and Lenk RP: DOTPLOT: Enhanced graphic matrix analysis of nucleic acid and protein
sequences. Proc Natl Acad Sci USA 78:7665, 1981.
Mayhew,S.G.and Ludwig,M.L.1995.Flavodoxin and electron-transferring flavoproteins.Academic
Press,Newyork,NY
Molecular Operating environment (MOE)V. 11, 2005, Chemical Computing group, Motreal,
Canada, http://www.chemcomp.com.
Nikolich, M., G. Hong, N. Shoemaker, and A. A. Salyers. 1994. Evidence that conjugal transfer of
a tetracycline resistance gene (tetQ) has occurred very recently between the normal
microflora of animals and the normal microflora of animals. Appl. Environ. Microbiol. 60:
3255-3260.
Osborne, C., Chen ,L.M., Matthews, R.G. 1991
Isolation, cloning, mapping, and nucleotide sequencing of the gene encoding flavodoxin in
Escherichia coli.J Bacteriol.;173:1729-37.
Robots, F., Roberts, C.N,. Johnson, J.J., et al. 1998, Evidence for shikomate pathway in
apicomplexon parasites, Nature, 393: 301-5.
44
Roberts, C.W., Roberts, F., Lyons, R.E.,Etc 2002., The shikimate pathway and its branches in
apicomplexan parasites.J Infect Dis.185 .1:S25-36.
Romero, A., Caldeira, J., Legall, J., Moura, I., Moura, J.J., Romao, M.J.,1996 . Crystal structure of
flavodoxin from Desulfovibrio desulfuricans ATCC 27774 in two oxidation states.Eur J
Biochem. 239:190-6.
Sali,A., Potterton, L., Yuan, F., van ,Vlijmen, H., Karplus, M. 1995. Evaluation of comparative
protein modeling by MODELLER.Proteins. 23,318-26.
Shiesh, S.C., Sheu, B.S., Yang, H.B., Tsao, H.J., Lin, X.Z. 2000. Serologic response to lowermolecular-weight proteins of H. pylori is related to clinical outcome of H. pylori infection in
Taiwan. Dig Dis Sci.; 45:781-8.
Sumita,T.,and Ramadevi,S.2004. In salico gene Identification and homology modeling of
chorismate synthase in clostridium difficle.Online Journal of Bioinformatics.5:129-131.144350.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994 , CLUSTAL W.Nucleic Acids Res.22:4673-80.
Tomb ,J.F., White, O., Kerlavage, A.R., 1997 The complete genome sequence of the gastric
pathogen Helicobacter pylori.Nature 388:539-47.
TIGR microbial databases, 2005. http://www.tigr.org/tdb/mdb/mdbinprogress.html#codes
Wotherspoon,A.C.,Doglioni,T.C.,Pan,L.,Moschini,A.,de Boni,M.,and Isaacson,P.G.1993.regression
of primary low- grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type
after eradication of Helicobacter pyroli.
Lancet 342:575-7.
.
45
Index Figures (Click to enlarge)
46
Index Table. 2. Ramachandran plot statistics
Description
Residues in most
favoured regions
Residues in
additional allowed
regions
Residues in
generously allowed
regions
Residues in
disallowed regions
End residues
(excl.Gly & pro)
Glycine residues
No.of residues
%
61
79.2
14
18.2
0
0.0
2
2.6
222
39
Proline residues
14
Total residues
352
100
©1996-2007 All Rights Reserved. Online Journal of Bioinformatics . You may not store these pages in any form except for your own personal
use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way
besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJB
publications. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors.
This journal satisfies the refereeing requirements (DEST) for the Higher Education Research Data Collection (Australia). Linking:To link to this
page or any pages linking to this page you must link directly to this page only here rather than put up your own page.
47