NADH-quinone oxidoreductase subunit B

Helicobacter pylori

Set-seq 5
Irving Flores Reg. Nr: 900623-241-050
Sergio Landeo Reg Nr: 820604-499-130

INTRODUCTION

Helicobacter pylor1 is a micro-aerophilic, Gram-negative, slowgrowing, spiral-shaped and

flagellated organism. H. pylori is probably the most common chronic bacterial infection of
humans, present in almost half of the world population due to its survival ability at acidic pH and
for its high prevalent presence in the gastric environment. Sequence analysis indicates that H.
pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and
modification. One of the systems preserved is the H +-translocating NADHquinone
oxidoreductase (Complex I) 2 which is found in the respiratory chains Complex I (also known as
NDH-1) which is generally is made up of 14 protein subunits; the genes that encode these
subunits are arranged in the same order in the different NDH-1 operons.
The 14 subunits of bacterial Complex I carry all the redox centres and structural elements
necessary for full enzymatic activity. However, the role of individual subunits in the enzymatic
activity and the identity of the redox center to which they bind (if they do) are not yet known with
certainty. An exception is the NQO1 (51 kDa) subunit (figure 1) which contains the binding sites
for the substrate, NADH, and for FMN and the tetranuclear FeS cluster N-3 and. The NQO2 (24
kDa) subunit is probably also involved in the binding and oxidation of NADH.
Sequencing of the entire H. pylori genome revealed the presence of an NDH-1 operon that is
ordinary except for the absence of genes that encode the NQO1 and NQO2 subunit3 . So, the
complex 1 of H. pylori is a NADH quinone reductase that cant oxidize NADH, accepting
electrons from other sources like quinones.

Figure 1:The NDH-1 operon of Helicobacter pylori, and its relation to the homologous gene cluster in Thermus thermophilus [9].
The latter is given as an example of a bacterial NDH-1 operon that contains only the structural genes and lacks additional ORFs.

The subunit in study of this complex, the subunit B which is the ortholog of the mitochondrial
PSST, is one of the seven subunit that form the periphereal arm of the NADH-Quinone
reductase complex in bacterias, and is fundamental for the electron translocation and assembly
of the structure.
An understanding of the particularities of such important complex for Helicobacter viability could
provide new target point for treatment through the search of, as example, possible inhibitor. Is
therefore a fortunate event having being able to recover this important data from the laboratory
accident.

METHODS
Identification of sequence
The result of sequencing was received as two FASTA files, with 21 forward and 20 reverse
reads respectively. For the assembling, both files were combined and uploaded to GALAXY
platform for using CAP3 greedy EST assembler, the consensus sequences were copy from the
output to reconstruct the nucleotide sequence. Then, the protein sequence was found using
ORF finder from Sequence Manipulation Suit.
To identify our gene, organism and protein we performed in NCBI web page a blastn search
against nucleotide collection (nr/nt) and a blastX search using BLOSUM 62 matrix. The protein
sequence was also analyzed for transmembrane domains with TMHMM, for signal peptide
using signalP and SOSUI and the protein domain was then identified using Simple Modular
Architecture Research Tool (SMART) and selects the Pfam domains in the search.
Modeling of protein
With the protein sequence, a Sequence Similarity Modbase Search was performed using the
default settings. After the search, two matches were found and a reliable model from them was
chosen based on the quality criteria contained in the model information of the target region,
template region, sequence identity, E-value and MPQS. With the coordinate file option, the pdb
file of the reliable target protein model was obtained. The Template PDB Code active link was
used to gather information related to the most important features of the experimentally obtained
structures used in Modbase to create the model. Also the Template PDB Code is useful to
know which subunit or chain of the complex or group of proteins were used to create the target
model protein. In our case, a subunit sequence was extracted from the complete template pdb
file by cutting only the subunit data that PDBViewer uses to create the 3D structure due to
model-subunit comparison. The target protein model pdb file and the subunit template pdb file
were uploaded to PDBViewer. Toolbar Fit- Iterative Magic Fit was performed on the structures
selecting Backbone atoms only and the layers involved (model-template) in the RMS & Auto Fit
options. Toolbar-Wind, Alingment, Layers info, Ramachandran Plot were used to visualize and
find snf identified conserved regions of the model protein. To evaluate the overall model quality
and local model quality, the target protein model pdb file was uploaded to ProSA-web Protein
Structure Analysis and then analysed.
Phylogenetic analysis
For making a phylogenetic tree once knowing the organism to which our protein belongs, a set
of related sequences was downloaded doing blastX searches (BLOSUM62) while narrowing the
search adjusting the search set to our organisms of interest. With this, a multiple sequence
alignment was performed using CLUSTALW (default setting) and a phylogenetic tree was made
using CLUSTALX 2.1 setting the bootstrapt labes on nodes and the number of bootstrap trials
in 1000. The result tree was then rooted using Treeview 1.6.6 with a sequence specifically
selected to be an outgroup.

RESULTS AND DISCUSSIONS

Identification of sequence.
The FASTA files were assembled in one contig of 482bp, with a coverage of 10, 27. As not all
the reads have the same length, the coverage was calculated using the average length of
reads. The coverage calculated is high and just one out of 41 sequences was not used (the
longer one). So, we received a high quality data.
Assembled sequence:
ATGCAACAAGCACCAGTTGTTCTAAGCACTTTGGATAAATTATTGAATTGGGGGCGTTCTAATTCGCTTTGGCC
TTTGACTTACGGCTTGGCGTGTTGCGCGATTGAGATGATGGCGACAGGGGGTTCAAGGTTTGATTTTGACCGG
TTTGGCACGATTTTTAGAGCTAGCCCTAGGCAATCTGATGTGATGATCATCGCTGGCACGCTCACTAAAAAACA
TGCCGAATTTATGCGCAGGCTTTATGATCAAATGCCTGAGCCTAAATGGGTGATTTCTATGGGGAGTTGCGCTA
ACACGGGCGGGATGTTTAACACTTATGCGACCGTTCAAGGAGCGGATAGGGTAGTTCCTGTGGATATTTATTT
GCCCGGTTGCGCGCCGCGTCCAGAGACTTTACAATACGCTCTTATGGTTTTGCAAGATAAAATCAGACGCTCT
AAAGCGATCAAACAAGACGCTCCTAAAAGGTTGGTGTGA

(40) 120.76
=
=
= 10.60

482

The ORF Finder in Sequence Manipulation Suit found a protein sequence in the first frame of
the direct strand, the sequence found was:
MQQAPVVLSTLDKLLNWGRSNSLWPLTYGLACCAIEMMATGGSRFDFDRFGTIFRASPRQSDVMIIAG
TLTKKHAEFMRRLYDQMPEPKWVISMGSCANTGGMFNTYATVQGADRVVPVDIYLPGCAPRPETLQY
ALMVLQDKIRRSKAIKQDAPKRLV

After performing the blastn search, we found a match with 100% identity with a gene in
Helicobacter pylori strain Shi470. The Query coverage was 100%, the max score and the total
score were 887, and the locus tag "HPSH_06535".
The blastn performed at NCBI identify the gene as NADH-quinone oxidoreductase subunit B
with 100% of identity, E-value of 2e-114, query coverage of 99% and a total and Max score of
333.Moreover, the accession number of the gene found is WP_001183511.1.
When uploaded our protein sequence into SMART, we
found a match with a Oxidored_q6 pfam domain with a
position 31-140 and a E-value 4.7E-25(HMMER3) (figure 2).
The uniprot domain identifier founded is NUOB-HELPH,
with this identifier we were able to retrieve from UNIPROT
information regarding functions, taxonomy, subcellular
location, interaction, family and domains.
Figure 2, domain found.

Our protein sequence gives no indication of having transmembrane helices in TMHMM or signal
peptide in signalP and SOSUI. The absence of transmembrane helices was an expected result
given that our protein is a subunit of the peripheral arm of the NADH-quinone oxidoreductase
complex, the complex 1 of the respiratory chain of Helicobacter pylori.

Protein modeling.
The model obtained from the Modbase compared a 2-158 target region from a 159 protein
length to a template region 15-170 of an experimental obtained Nqo6-NADH-quinone
oxidoreductase subunit 6-Thermus thermophilus (Template PDB Code:3I9V6) with 61%
sequence identity, E-Value 0, GA341 1.00 as important quality criteria to consider it as a reliable
model.
The modbase-model_5 pdb file (NADH-quinone oxidoreductase subunit B-Helicobacter Pylori
model) was uploaded in conjunction with the 39IV6Template pdb file (NADH-quinone
oxidoreductase subunit Thermus Thermophilus) to PDBViewer to perform an interative magic
fit obtaining a value of 61.0 in the alignment between both sequences visible in the Alignment
wind. A RMS value of 0.57 A regarding how good modbase-model_5 was superimposed to
39IV6Template was achieved Apendix 3
Homologues from bacterial and
archaeal species have shown to
contain 8 conserved acidic amino
acids including 3 glutamates, 5
aspartates, residues essential for
electron transfer and assembl 4 .
The conserved amino acids of the
NADH:
Ubiquinone
Oxidoreductase
Complex
ISubunitB present in multiple
species were also evident in an
alignment in ClustalX that was
performed using 13 sequences
chosen by its taxonomical relation
(appendix 1). Equivalent position
of acidic amino acids were
identified using the alignment and
located in the protein model
structure modbase-model_5 as
Glu 36, Asp 46, Asp 62, Asp 83,
Glu 87, Asp 114, Asp 120, Glu
131.

Figure 2. model of our protein and the conserved residues.

It is expected that Glu 36, Asp 46 and Asp 62 are essential for electron transfer. Glu 36 might
have the same proposed role as Glu49 in Nqo64 (T. thermophilus) as the acidic amino acid that
is protonated whereas FeS Cluster N2 is oxidized and provides the proton to the close subunit
Nqo4 that can deliver it to the quinone or to a proton channel formed by the hydrophobic subunit
L organized as the studied architecture of the hydrophilic domain of T.thermophilus complex I
(figure 3).

Figure 3. Hydrophilic domain (Nqo1-Nqo15,3I9V) and the alpha-helical model for the membrane domain
(Nqo10-Nqo14). Fe-S clusters are shown as red and yellow spheres, and FMN as magenta spheres.

The Ramachandran plot of model shows both almost all residue in the favorable zones, besides
G residues just a couple of A and V are on the boundaries of the energetically favorable zones
(appendix 2). Overall model quality result in a Z-score of -4.43 and acceptable values for local
model quality were obtained after the residue 40 approximately (figure 4).

Figure 4. Overal model quality and local model quality.

Phylogenetic analysis.
From the domain architecture analysis in SMART, 7307 proteins are listed as having a domain
organization matching the protein sequence. This proteins are present in all kingdoms: Archae
(644), Bacteria(5596), Eukaryota(993) and undefined superkingdom(74). So the sequences
used to build the phylogenetic tree are listed in the appendix 2. The sequences were chosen to
include not only representative information from different bacteria phylum, but also to include an
Euryarchaeota sequence to root the tree and several representative of the proteobacteria
phylum to which Helicobacter pylori belongs.

Figure 5, phylogram.

In the phylogram (figure 5), which also include representatives of phylum Acidobacteria
(Chloracidobacterium thermophilum), Firmicutes (Bacillales), Deincoccus-Thermus (Thermus
thermophilus), Actinobacteria (Streptomyces somaliensis) and Spirochaetes (Leptonema illini), it
is shown that proteobacteria are organized in two different cluster. Helicobacter pylori is
clustered with the representatives of the Order Campylobacterales, and the representatives of
the Order Enterobacterales are in clustered separately.This result, althoug seems to be
unespected was known posibility given that many examples of H. pylori proteins have greater
sequence similarity to those found in non proteobacteria 1 . From the phylogram obtained can
be seen that this pattern is also present in the other chosen members of Campylobacterales
Order and could be due, given the ancient divergence shown in this phylogram, of an early
divergence of this group from the lineage that led to, at least, Enterobacterales Order.

It is also shown that, among this orthologs of NADH-quinone oxidoreductase subunit B, the
distances shown in the phylogram are consistent with the taxonomic classification of the chosen
representative of Proteobaceria Phylum. Among the Campylobacter representatives, the
divergence of both genre of Helicobacter is the most recent event followed by the divergence
from the other families of this Order; and among the representatives of Enterobacterales the
divergence showed is also recent. Moreover, the bootstrap values in these two clusters are high
enough to have confidence in classification obtained.

REFERENCES

1.- Tomb JF., et. al. (1997) The complete genome sequence of the gastric pathogen
Helicobacter pylori. Nature 388, 539547
2.- Takao Y. (1998) Procaryotic complex I (NDH-1), an overview. Biochimica et Biophysica Acta
(BBA) - Bioenergetics 1364, 125-133

3._ Moshe Finel (1998) Does Nadh play a central role in energy metabolism in Helicobacter
pylori? Trends in Biochemical Sciences 23, 412-414
4._ Dirk Flemming, et. al- (2006) Catalytic Importance of Acidic Amino Acids on Subunit NuoB
of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I).Journal of biological
chemistry. 25, 24781- 24789
5.- Sazanov, L. A. and Hinchliffe, P. (2006) Structure of the Hydrophilic Domain of Respiratory
Complex I from Thermus thermophiles. Science 311, 1430-1436

APENDIX 1

APENDIX 2

Target protein model

Template-Model

Template protein model

Template(yellow)-Model(green)

APENDIX 3
Species

Max
score

Total
score

Query
cover

E value

Ident.

Accession

E. coli

179

179

89%

5e-55

53%

CDL36829.1

Synechocystis

178

178

87%

1e-56

56%

WP_010872906.1

H. canis

291

291

97%

6e-98

88%

WP_023930734.1

Salmonella enterica

178

178

89%

4e-55

53%

WP_045722202.1

Yersinia pestis

172

172

88%

8e-54

52%

WP_016681330.1

Bacillales (multispecies)

212

212

88%

2e-67

67%

WP_029100362.1

Streptomyces somaliensis

198

198

89%

2e-62

62%

WP_010470882.1

Chloracidobacterium
thermophilum

219

219

94%

5e-72

64%

AEP12619.1

Wolinella succinogenes

286

286

97%

3e-100

84%

WP_011138413.1

Campylobacter lari

286

286

100%

2e-98

80%

WP_012662142.1

Methanocaldococcus
villosus

105

105

70%

3e-29

38%

WP_004592815.1

Thermus thermophilus

213

213

91%

8e-71

65%

AEG32731.1

Leptonema illini

174

174

93%

3e-54

50%

WP_040919347.1