bioinformatics structural prediction of fmta gene from ten various species of bacteria and...

Structural Prediction through sequence analysis using bioinformatics tools:

Prediction of the most distant species of fmtA protein from a variety of 10 species

using multiple sequence analysis

Nemerisza I. ARAN, Relyando DE FIESTA, Dane Nathalie T. MIRANDA and Justine

Rose F. SANTOS

Chemical Engineering Department, Technological Institute of the Philippines, Manila

1001, Philippines

Abstract

Various bacteria from different species that contains the fmtA protein was obtained using

the NCBI genbank. This paper contains fmtA protein sequences from gram-positive and

gram-negative bacteria that has different functions and characteristics. With the use of

protscale, the researchers were able to determine the molecular weight and chemical

formula of each species. B. pseudomallei with a molecular weight of 82492.3 Da is

considered to be the heaviest among the remaining bacteria and S. aureus with a

molecular weight of 46067.4 Da being the lightest. All the species chosen gave partial

hydrophobic results based on the Protscale result and all being hydrophilic in the

protparam result, G. lozoyensis having the highest number of major peaks and S.

epedermidis being the lowest. Data of motifs that matched in each sequence was given

by MotifScan, only two bacteria gave no match and the rest gave possible results. Two

domains were identified, the -lactamase and TonB, some shared the same domain.

Proteases and chemicals that are capable of digesting the given bacteria was given by

Peptide cutter.

Secondary structure analysis using Psipred determined how many -helices, -sheets

and random coils each fmtA protein have. S. sanguinis has the most -helix, B.

pseudomallei and H. alvei tied in having the most number of -sheets and Y. regensburgei

having the most number of random coils.

It was found that B. pseudomallei is farthest from the reference ancestral fmtA protein

with a distance of 19.57, and was used in determining the tertiary and quaternary

structure.

Keywords: fmtA protein, protein structure, multiple sequence alignment, bacteria,

bioinformatics

Introduction

Protein sequences emerging from genome sequencing projects are of greatest value to

medicine and biology if their structure and function can be identified. With the growing

number of annotate sequences, association of a new sequence to a protein of known

structure can be a significant step towards the identification of its biological role. Simple

sequence search methods such as FASTA (Pearson and Lipman, 1988) or NCBI readily

identify close homologs of protein sequences.

Multiple Sequence Alignment improves the detection of distantly related homologous

protein. ClustalW and Tcoffee are the most common procedure when doing multiple

sequence alignment, the sequences were grouped according to their similarities into a

tree (hierarchical cluster analysis). Starting with the most similar pairs, all the sequences

are aligned stepwise to each other using the dynamic programming method. The aligned

sequences are output as well as the cluster analysis, but these procedures normally do

not include any statistical analysis of the significance of the alignment.

The fmtA gene was identified to be a methicillin resistance factor in Staphylococcus

aureus. Inactivation of fmtA leads to increased sensitivity of methicillin-resistant S.aureus

strains (MRSA) to Triton X-100 and -lactams and decreases the level of highly cross-

linked peptidoglycan (PG) (Komatsuzawa et al). Ten sequences of fmtA gene from

different bacterial species were entered to a protein structure identifier database to

compare the primary to secondary structure of fmtA genes from each given species of

bacteria. Then, having the most distant specie in predicting the structure of tertiary and

quaternary structure.

Methods

Control dataset

A dataset of fmtA protein from different bacterial species obtained from NCBI GenBank

(http://www.ncbi.nlm.nih.gov/) is listed here: EEV75348.1, EFH09490.1, EHL01514.1,

WP_009496590.1, YP_001066210, WP_004191820.1, WP_002496510.1, EHM51293.1,

EHL97443.1, EHM40511.1. The protein sequences of this 10 accession numbers were

used throughout this paper.

Physico-chemical properties. For the computation of physico-chemical properties,

ProtParam (http://web.expasy.org/protparam/) was used. The computed parameters

include the molecular weight, theoretical pI, amino acid composition, atomic composition,

estimated half-life and grand average of hydropathicity (GRAVY). Extinction coefficient,

absorbance and aliphatic index was calculated using the following formula

E(Prot) = Numb(Tyr)*Ext(Tyr) + Numb(Trp)*Ext(Trp) + Numb(Cystine)*Ext(Cystine)

Absorb(Prot) = E(Prot) / Molecular_weight

Aliphatic index = X(Ala) + a * X(Val) + b * [ X(Ile) + X(Leu) ]

Identifying Protein Domains. InterProScan (www.ebi.ac.uk/InterProScan/) combines

different protein signature recognition methods and allows the comparison of a certain

sequence from the InterPro, a domain database that includes most of the major domain

collections available online.

Motifs determination. Motif (http://myhits.isb-sib.ch/cgi-bin/motif_scan) scanning means

finding all known motifs that occur in a sequence. In using this database, the result must

be filtered and shown using PROSITE profiles.

Transmembrane Segment Prediction.

THMM (http://www.cbs.dtu.dk/services/TMHMM/) is used to predict transmembrane

segments in protein. It also tells about the portion of proteins that are probably inside and

outside the cell.

Locating Coiled-coil Region. In determining the coiled-coil region of the given protein

structure, http://ch.embnet.org/software/COILS_form.html. COILS is a program that

compares a sequence to a database of known parallel two-stranded coiled-coils and

derives a similarity score.

Hydrophobicity prediction. In computing ang representing the profile produced by any

amino acid scale on a selected protein, ProtScale (http://web.expasy.org/protscale/) was

used. It is a two-dimensional plot wherein the hydrophobicity of a protein is given account

to. This concerns major and minor peaks that are responsible in the determination of the

hydrophobic site. In analyzing the plot, the number of hydrophobic and hydrophilic peaks

was counted at the score of ()1st respectively.

Detecting PROSITE signature matches. To detect which functional group or protein will

help in increasing the functional diversity of proteome, a trusted protein database was

used (http://prosite.expasy.org/scanprosite/). ScanProsite is a web-based tool in

determining which prosite pattern a certain sequence is located. It is also designed for

checking if other proteins contain the same sequence.

Predicting cleavage sites. PeptideCutter (http://web.expasy.org/peptide_cutter/) was

used in predicting potential cleavage sites cleaved by proteases or chemicals in a given

protein sequence. This tool can be helpful in determining whether the chosen protein can

interact with the available enzyme in the database. Enzymes can be chosen all at once,

and can also be chosen one at a time depending on how the user wants in to be.

Prediction of the secondary structure

PSIPRED (www.bioinf.cs.ucl.ac.uk/psipred/) is a popular structure prediction method to

accurately predict the secondary structure of any protein. It can say how many alpha helix

and beta sheets are in there in a protein structure.

Prediction of the tertiary structure

Dihedral angles between C-C (, psi) and N-C (, phi) of amino acid residues and

empirical distribution of data points in a protein structure are determined using Rampage

(http://mordred.bioc.cam.ac.uk/~rapper/rampage.php).

Prediction of the quaternary structure

SWISS-MODEL (http://swissmodel.expasy.org/) is a structural bioinformatics web-

server dedicated to homology modeling of protein 3D structures. Homology modeling is

currently the most accurate method to generate reliable three-dimensional protein

structure models and is routinely used in many practical applications.

Results and Dscussion

Primary sequence analysis

[Sporosarcina newyorkensis]

Number of amino acids 625

MW 72531.6 Da GRAVY: -0.1

Instability Index 43.79, protein is unstable Aliphatic Index 99.28

Extinction Coefficients 113110 Absorbance 1.559

[Burkholderia pseudomallei 1106a]


MW 82434.2 Da

Instability Index 31.03, protein is stable Aliphatic Index 75.34


[Streptococcus sanguinis]


MW 67379.2 Da GRAVY: -0.235

Instability Index 26.40, protein is stable Aliphatic Inedx 88.26


[Staphylococcus aureus A8115]


MW 46067.4 Da GRAVY: -0.561



[Roseomonas cervicalis ATCC 49957]


MW 79740.9 Da GRAVY: -0.312



[Glarea lozoyensis 74030]


MW 56389.9 Da GRAVY: -0.127



[Staphylococcus epidermidis]


MW 46620.6 Da GRAVY: -0.584



[Yokenella regensburgei ATCC 43003]


MW 81182.9 Da GRAVY: -0.521



[Acetobacteraceae bacterium AT-5844]


MW 79079.1 Da GRAVY: -0.327



[Hafnia alvei ATCC 51873]


MW 80466.0 Da GRAVY: -0.531



Fig.1. Physico-chemical properties of the fmtA protein in

different bacteria.

Identifying Protein Domains

Fig.2a. Resulting domain for gram-positive bacteria

Fig.2b. Resulting domain for gram-negative bacteria

Matches E-value

[Staphylococcus aureus A8115]

Beta-lactamase

Beta-lactamase PENICILLIN-BINDING PROTEIN

transmembrane_regions

signal-peptide

5.0E-62 [88-383] T

5.0E-62 [88-383] T 5.9E-33 [15-295] T

-1.0 [9-27] ?

-1.0 [1-31] ?

[Roseomonas cervicalis

ATCC 49957]

G3DSA:2.40.170.20

TonB_dep_Rec TAT

G3DSA:2.170.130.10

Plug PTHR32552

PTHR32552:SF0

SSF56935

0.0 [195-731] T

5.3000000000000115E-28 [487-730] T 0.0 [1-40] T

4.099999999813685E-38 [38-192] T

4.400000000000006E-22 [82-181] T 0.0 [7-731] T 0.0 [7-731] T

0.0 [55-731] T

[Glarea lozoyensis 74030]

Beta-lactamase

G3DSA:3.40.710.10 PBP_transp_fold

PTHR22935

1.0999999999999873E-48 [3-354] T

2.9999999998641766E-60 [3-358] T 2.9999875044570637E-61 [5-369] T 3.4999946686394883E-40 [1-337] T

[Sporosarcina newyorkensis]

Beta-lactamase

G3DSA:3.40.710.10 PBP_transp_fold

PTHR22935

1.2000000000000005E-47 [68-379] T

9.099999999558015E-61 [56-393] T 1.2000011745813432E-59 [51-403] T 3.9999854413940615E-41 [68-378] T

[Burkholderia pseudomallei

1106a ]

Beta-lactamase G3DSA:3.40.710.10

PBP_transp_fold PTHR22935

1.2000000000000005E-47 [68-379] T 9.099999999558015E-61 [56-393] T

1.2000011745813432E-59 [51-403] T 3.9999854413940615E-41 [68-378] T

[Streptococcus sanguinis]

Beta-lactamase G3DSA:3.40.710.10

PBP_transp_fold

PTHR22935

3.999999999999978E-51 [56-355] T 2.0999999998359858E-66 [57-355] T

7.40000780398351E-66 [31-371] T

3.800006970935884E-41 [36-352] T

[Staphylococcus

epidermidis]

Beta-lactamase no description

beta-lactamase/transpeptidase-like

PENICILLIN-BINDING PROTEIN signal-peptide

transmembrane_regions

3.9E-49 [87-368] T 1.0E-54 [70-368] T 4.5E-68 [62-387] T

3.9E-29 [73-386] T -1.0 [1-26] ? -1.0 [9-29] ?

[Yokenella regensburgei

ATCC 43003]

G3DSA:2.40.170.20 TonB_dep_Rec TonB-siderophor

TONB_DEPENDENT_REC_1 TONB_DEPENDENT_REC_2

G3DSA:2.170.130.10 Plug

0.0 [197-733] T 1.6999999999999923E-27 [489-732] T

0.0 [79-733] T

0.0 [1-48] T 0.0 [716-733] T

3.099999999755361E-40 [32-193] T 1.3000000000000007E-22 [78-182] T

[Acetobacteraceae bacterium AT-5844]

G3DSA:2.40.170.20 TonB_dep_Rec

TonB-siderophor G3DSA:2.170.130.10

Plug PTHR32552

PTHR32552:SF0 SSF56935

0.0 [188-720] T 1.9000000000000044E-32 [494-719] T

9.900000000000002E-130 [76-718] T 8.800000001009721E-38 [53-182] T

2.1000000000000035E-21 [75-174] T 0.0 [39-720] T

0.0 [39-720] T 0.0 [29-720] T

[Hafnia alvei ATCC 51873]

G3DSA:2.40.170.20 TonB_dep_Rec TonB-siderophor

TONB_DEPENDENT_REC_2 G3DSA:2.170.130.10

Plug

PTHR32552 PTHR32552:SF0

SSF56935

0.0 [196-728] T 1.9999999999999946E-25 [489-727] T

0.0 [80-728] T

0.0 [711-728] T 1.0E-40 [33-192] T

5.099999999999984E-24 [79-181] T

0.0 [52-728] T 0.0 [52-728] T 0.0 [52-728] T

Table 1. Summary of matches and E-value from domains obtained using Interproscan

The functions of unknown proteins can be recognized by matching its motif with those of

the known ones using InterProscan a tool from Expasy. FmtA protein in S. aureus, S.

newyorkensis, S. sanguinis, S. epedermidis have the same domains of Beta-lactamase

related and Beta-lactamase/transpeptidase-like. Beta-lactamase catalyses the opening

and hydrolysis of the beta-lactam ring of beta-lactam antibiotics such as penicillins and

cephalosporins. Most of these antibiotics work by preventing biosynthesis of the bacterial

cell wall. The possibility of Staphylococcal and Streptococcal bacteria to have Beta-

lactamase as the primary domain is that these kind of organisms are capable of resisting

many forms of important antibiotics. G. lozoyensis also share this domain and has

Peptidase S12, Pab87-related, C-terminal as another domain. The common

characteristic of the five (5) bacteria is that they are all gram-positive.

As for fmtA in R. cervicalis, B. pseudomallei, Y. regensburgei, A. bacterium and H. alvei

share the same domains for TonB-dependent receptor, beta-barrel, siderophore receptor

and plug. An extensional domain of TonB-dependent receptor, conserved site for both Y.

regensgurgei and H. alvei. TonB box, conserved site as another domain in H. alvei.

TonB is responsible in interacting with the outer membrane receptor proteins. These

proteins carry out high-affinity binding energy-dependent uptake of specific substrates

into the periplasmic space. The periplasmic space is the space between the cell wall and

the cell membranes. Bacteria that has TonB as domain are all gram-negative, just like R.

cervicalis, B. pseudomallei, Y. regensgurgei, A. bacterium and H. alvei.

Motif information Status Position Raw-score N-score E-value

Fmta [Staphylococcus aureus A8115] Big-1 (bacterial Ig-like domain 1) domain

BIG1 Weak match 1-9 33 4.128 1.6e+03

Ferric malleobactin receptor fmta

[Roseomonas cervicalis ATCC

49957]

NHL repeat profile

NHL Weak match 306-318 27 4.009 2.1e+03

Twin arginine translocation

TAT Weak match 1-40 858 7.882 0.28

Putative protein fmta [Glarea

lozoyensis 74030] No match

Fmta family protein [Sporosarcina

newyorkensis]

LDL-receptor class B repeat profile

LDLRB Weak match 617-625 153 5.329 99

Ferric malleobactin transporter

[Burkholderia pseudomallei 1106a ]

Alanine-rich region

ALA_RICH Strong match 27-83 48 9.353 0.0094

Fmta family protein [Streptococcus

sanguinis] No match

Fmta family protein [Staphylococcus

epidermidis]

Lysine-rich region

LYS_RICH Weak match 52-83 40 6.918 2.6

Bipartite nuclear localization signal profile

NLS_BP Weak match 52-66 3 3.000 2.1e+04

Putative ferric malleobactin receptor

fmta [Yokenella regensburgei ATCC

43003]

Threonine-rich region Weak match 67-136 38 6.929 2.5


fmta [Acetobacteraceae bacterium

AT-5844]

No match


fmta [Hafnia alvei ATCC 51873]

MVP (vault) repeat

MVP Weak match 61-114 446 5.366 91

Table 2. Corresponding motif for each bacteria; given extra information about the position, raw-score, N-score and E-value

Motifs determination.

Among the resulting motifs, only B. pseudomallei gave the strongest response with

regards to its corresponding motif, which is ALA_RICH, given the highest possible N-

score of 9.353. Both S.sanguinis and A. bacterium gave no match to any motif available

in the MotifScan database. All remaining bacteria gave a weak match, this means that

there is a probability that the given motifs are unsure and does not comply to each given

protein sequence.

E-value provides an estimation of the number of false positives. Among the weak

matches, NLS_BP with an E-value of 2.1x104 gave the lowest possibility of it being false

positive match to S. epidermidis. For having an E-value of 99, LDLRB has the highest

possibility of it being a false match to S. newyorkensis.

S. epidermidis is said to be a part of humans normal bacterial flora and is associated with

foreign infection. This cocci has low pathogenic potential for those who have strong

immune system. Having weak response from NLS_BP, which has a primary role in

describing a specific sequence within a protein that is responsible for the translocation

into the cell, there must be a possibility that the protein sequence of S. epidermidis

happens to be have few amino acids that are capable in transferring or infecting other life

forms.

S. newyorkensis is an endospore-forming bacteria capable of transferring some of its

DNA to a host. Endospores are commonly found on places where it can survive for a long

period of time, they can be found on soil and water. The function of LDLRB is to regulate

and maintain internal stability of cholesterol in mammalian cells. The difference between

S. newyorkensis and LDLRB is that one does not have to modify the endospores that it

forms and may let it lie dormant for a very long time, and the other has to check-up on the

cholesterol levels once in a while to make sure that everything is at equilibrium.

Transmembrane Segment Prediction

Table 3. Given the posterior probabilities of being on the inside or outside of the cell

Inside Transmembrane helix Outside

Staphylococcus aureus A8115 1 - 6 7 - 26 27 - 397

Roseomonas cervicalis ATCC 4995 1 - 731

Glarea lozoyensis 7403 1 -513

Sporosarcina newyorkensis

1 - 25

524 - 531

590 - 601

26 - 48

501 - 523

532 - 554

569 - 589

602 - 624

49 - 500

555 - 568

625 - 625

Burkholderia pseudomallei 1106a 1 - 753

Streptococcus sanguinis

1 - 4

481 - 492

553 - 563

5 - 24

458 - 480

493 - 515

530 - 552

564 - 586

25 - 457

516 - 529

587 - 592

Staphylococcus epidermidis 1 - 6 7 - 29 30 - 400

Yokenella regensburgei ATCC 43003 1 - 733

Acetobacteraceae bacterium AT-5844 1 - 720

Hafnia alvei ATCC 51873 1 - 728

In determining where the residue is on the cell, the TMHMM plots must be the source of

information and not the probabilities listed above, because the plot shows the location

and the data above shows only the prediction of the location if the transmembrane helix

is on the inside, outside or inside the membrane of the cell.

With respect to the resulting graph of each species, S. aureus, B. pseudomallei, A.

bacterium, H. alvei, Y. regensgurgei and S. epedermidis are inside the cell. G.

lozoyensis showed no TM helix. R. cervicalis is outside the cell. S. newyorkensis

showed inside positions at 25-50, 495-520, 525-550, 555-580. 600-625. Lastly, S.

sanguinis at the position of 1-25, 450-480, 485-510, 520-560, 570-592 showed to be

inside.

Determining the coiled-coil region

Fig 3. Number of coiled-coil regions for different species.

Coiled coils are built by two or more alpha-helices that wind around each other to form a

supercoil. In essence coiled coils are built of sequence elements of three and four

residues whose hydrophobicity pattern and residue composition is compatible with the

structure of amphipathic alpha-helices. S. newyorkensis having the most number of

coiled-coil region with a score of 8 and A. bacterium being the least with a score of 2.

Hydrophobicity prediction

Based on the GRAVY calculated using Protparam, species were considered hydrophilic

due to a very low score. In determining how hydrophobic and hydrophilic each species

are, the Protscale plot was used as the basis for analysis. Peaks above zero indicates

that the residue is hydrophobic, below zero is hydrophilic.

0 1 2 3 4 5 6 7 8

NUMBER OF COILED COIL REGION

Hafnia alvei ATCC 51873 Acetobacteraceae bacterium AT-5844 Yokenella regensburgei ATCC 43003Staphylococcus epidermidis Streptococcus sanguinis Burkholderia pseudomallei 305Sporosarcina newyorkensis Glarea lozoyensis 74030 Roseomonas cervicalis ATCC 49957Staphylococcus aureus A8115

Fig 4. Prediction of the number of major peaks having ()1 as the base point in the

ProtScale plot.

According to the figure, G. lozoyensis with 13 major peaks has the most hydrophobic

residue and S. epedermidis with 4 major peaks being the least. The hydrophilic peaks are

more in number than the hydrophobic peaks. H. alvei with 20 peaks is considered the

most hydrophilic among all species. Having 12 peaks, S. aureus, S. epedermidis and S.

sanguinis are the least hydrophilic among all the species.

Determining the post-translational modification

Some PTMs of the given bacteria are identical with each other and some has a unique

modification. Figure A shows the PTMs for each bacterium, it was arranged according to

similarity of their modifications. Only two bacteria have active sites namely, Y.

regensburgei ATCC 43003 and H. alvei ATCC 51873. Active sites for Y. regensburgei

ATCC 43003 and H. alvei ATCC 51873 are TonB-dependent receptor proteins signatures

1 and 2 (TonB-DRPS 1 & 2) and TonB-dependent receptor proteins signature 2 (TonB-

DRPS 2), respectively. Without TonB, receptors attach their substrates even though it

does not have an active transport. Active transport is important in moving ions through

5

9

13

912

8

4

9 96

12

1714 14

17

12 12

18 1720

hydrophobic peaks hydrophilic peaks

membranes contrary to their electrochemical gradient. A summarized table below is

provided to show how many patterns in the sequence of bacteria are identical in the

prosite database.

Fig. 5. The chart shows the post-translations modification for each bacterium. The name

of the bacteria is placed outside the circle. Similar PTMs for the 10 bacteria are located

at the center. PTMs positioned in between two colors correspond to two bacteria. While

PTMs found inside the segment of the circle is a unique modifier for that specific bacteria.

Gla

rea

lozoyensis 7

40

3

CK2, PKC

Phosphorylation

N-glycosylation

N-myristoylation

leucine

zipper

pattern

TonB-DRPS

1 & 2

TonB-

DRPS 2

Detecting PROSITE signature matches

Table 4. The table shows the number of hits by pattern in a sequence for each bacterium

Protein kinases are responsible for phosphorylation; it is a kind of enzyme that catalyzes

transfer of phosphate group from ATP to substrates. They are usually used in transmitting

signals and controlling processes in cells. Its known function is to modify the activities of

proteins. Some of the functions of phosphorylation are regulating the functions of proteins

by formation and disturbance of protein-protein surfaces and by stimulating

conformational changes. Due to the reversibility of phosphorylation, it permits cell to

respond to stimuli making it to be perfect tool in signal transduction. Individual functions

for the different types of phosphorylation that was used in modifying the given bacteria

are also discussed. Phosphorylation of tyrosine residues controls the enzymatic activity

of the protein and generates binding sites for downstream signaling proteins. Casein

kinase (II) is concern mainly on regulating cellular processes. They are self-regulating on

cyclic nucleotides and calcium. Protein kinase C is used for phosphorylation of serine or

threonine residues adjacent to the C-terminal basic residue. It improves enzyme-

catalyzed reaction and substrate concentration of phosphorylation reaction.

fmtA Sequence Hits by pattern

Staphylococcus aureus A8115 16 out of 397 amino acids

Roseomonas cervicalis ATCC 4995 47 out of 731 amino acids

Glarea lozoyensis 7403 31 out of 513 amino acids

Sporosarcina newyorkensis 24 out of 625 amino acids

Burkholderia pseudomallei 1106a 35 out of 753 amino acids

Streptococcus sanguinis 23 out of 592 amino acids

Staphylococcus epidermidis 22 out of 400 amino acids

Yokenella regensburgei ATCC 43003 48 out of 733 amino acids

Acetobacteraceae bacterium AT-5844 42 out of 720 amino acids

Hafnia alvei ATCC 51873 56 out of 728 amino acids

N-myristoylation serves as a conformational localization switch, the conformational

changes of protein has a major effect in the availability of the handle for membrane

attachment. It also increases the hydrophobicity and affinity of membranes due to

myristoyl group which attaches to the N-terminal amino acid of polypeptide. N-

myristoyltransferase (NMT) is the enzyme used in catalyzing the modification.

N-glycosylation improves the functional diversity of proteome. It confirms whether the

folded proteins are transferred to Golgi or not. N-linked glycoproteins contribute important

properties during protein folding, conformation, distribution, stability and activity.

Amidation improves the activity of peptides and lengthens its shell life. Together with N-

terminal acetylation, it lessens the overall charge of peptides causing the solubility to

decrease. Moreover, it enhances the resistance of peptides against enzymatic

degradation and increases its stability as they copy the native protein. Therefore,

amidation boosts the biological activity of peptide.

S. sanguinis has a unique modification called leucine zipper pattern. It is responsible for

binding DNA within the promoters of genes. It regulates gene expression in order to

develop complex organisms.

Predicting cleavage sites

Fig. 6. Number of possible cleavage sites obtained using PeptideCutter.

With respect to the number of cleavages, Proteinase K cleaved most of the amino acid in

a corresponding sequence for each bacteria. This enzyme preferentially cleaves at

aliphatic of aromatic amino acid such as Tyrosin, Phenylalanine, Tryptophan and

Histidine (Keil, 1992). Other function includes major role in the destruction of proteins in

cell lysates (tissue, cell culture cells) and for the release of nucleic acids. Since almost all

of the chosen sequence contain a lot of aromatic amino acid. Next on the list is

Thermolysin, this proteinase cleaves sites with bulky and aromatic residues Isolucine,

Leucine, Valine, Alanine, Methaionine and Phenylalanine (Keil, 1992).

Trypsin, being in in the middle, preferentially cleaves at Arg and Lys in position P1 with

higher rates for Arg especially at high pH (Keil, 1992). Hydroxylamine, with a low score

of one-digit number, is responsible in cleaving sites at Asn and Glu (Bornstein & Balian).

Lastly, Enterokinase, having no cleavage at all, is a serine protease that recognizes the

0

50

100

150

200

250

300

350

400

190

381

256

344372

317

205

363378

357

115

205

153

207221

183

116

194 205 183

59 63 47 5767 62 54 59 60 62

3 3 2 2 2 3 3 2 1 20 0 0 0 0 0 0 0 0 0

NO

. OF

CLE

AV

EGES

Proteinase K Thermolysin Trypsin Hydroxylamine Enterokinase

amino acid sequence -Asp-Asp-Asp-Asp-Lys-|-X (Roche) with a high specificity. The

enterokinase activates its natural substrate trypsinogen and releases trypsin by cleavage

at the C-terminal end of this sequence.

Together with enzyme enterokinase, Caspase1-10 is not intended for fmtA from all

bacteria except for Sporosarcina newyorkensis and Streptococcus sanguinis because

these bacterium can digest Caspase8. Enterokinase and Granzyme B enzymes has a

preference for cleaving which fmtA lacks, thats why these enzymes did not give possible

cleavage sites in the sequence. For Factor Xa, only Roseomonas cervicalis is applicable.

FmtA proteins from Roseomonas cervicalis, Burkholderia pseudomallei, Yokonella

regensburgei and Aceterobacteraceae bacterium can digest the enzyme Tobacco etch

virus protease. For Thrombin, only Roseomoinas cervicali gave a positive feedback.

Prediction of secondary structure

Table 4. Summary of results obtained using PSIPRED, indicated the number of -helices,

-sheets and random coils in the structure of each fmtA protein in different bacteria

-helix -sheet Random coils

[Staphylococcus aureus

A8115]

11

9 21

[Roseomonas cervicalis

ATCC 49957] 4 36 41

[Glarea lozoyensis 74030] 11 19 30

[Sporosarcina newyorkensis] 16 17 33

[Burkholderia pseudomallei

1106a] 4 38 42

[Streptococcus sanguinis] 15 17 32

[Staphylococcus

epidermidis] 11 9 21

[Yokenella regensburgei

ATCC 43003] 3 39 43

[Acetobacteraceae bacterium

AT-5844] 3 36 40

[Hafnia alvei ATCC 51873] 3 38 42

Secondary protein structure is the specific geometric shape caused by intramolecular

and intermolecular hydrogen bonding of amide groups. It composes of -helix, -sheets

and sometimes random coils.

Based on the Psipred result of 10 sequences, it is noticeable that the -sheets have a

higher number compared to the -helix. in the -helix structure, the "backbone" of the

peptide forms the inner part of the coil while the side chains extend outward from the coil.

-sheets have a greater number because not all amino acids favor the formation of the

-helix due to steric constraints of the R-groups. Amino acids such as A, D, E, I, L and M

favor the formation of -helices, whereas, G and P favor disruption of the helix. This is

particularly true for P since it is a pyrimidine based imino acid (HN=) whose structure

significantly restricts movement about the peptide bond in which it is present, thereby,

interfering with extension of the helix. Whereas an -helix is composed of a single linear

array of helically disposed amino acids, -sheets are composed of 2 or more different

regions of stretches of at least 5-10 amino acids.

Fig. 7. Multiple sequence alignment of fmtA. The colors of the letters correspond how good or bad the sequence identity.

Multiple sequence alignment is used to detect related proteins and to study the

relationship between the sequences. It has been clearly shown that using multiple

sequence alignments improve upon the detection of distantly related homologous

proteins.

Figure shows the multiple sequence of the entered sequence. Each of the sequence is

listed with their names and an alignment. The color of each letter tells how bad or good

the alignment is. Notice how the first and last part of the protein has a good sequence

identity. These proteins are conserved through evolution.

Fig. 8. Phylogeny of fmtA protein fromm different bacteria, it is divided into three

clusters: Distance were calculated by means of % identity.

The proteins in the alignment can be grouped according to different sequence features.

This allows the proteins to be further assorted into subgroups that are most closely

related to each other. If the protein is not well conserved, it indicates that it is more

evolutionary distant.

Sequences that are more related are closer together in the branch of the tree. Based on

the fig. 8 the ancestor (base point of zero) of the protein is closely related to S. aureus

and S. epidemidis. In contrast, B. pseudomallei are the most distant from ancestral

protein. B. pseudomallei is a Gram-negative bacteria pathogen that normally survives as

a saprophyte in soil and water, but is also capable of infecting most mammals and causing

serious infections resulting in the multifaceted disease melioidosis. Very little is known

about iron acquisition mechanisms in B. pseudomallei. The bacterium produces a

hydroxamate-type siderophore, malleobactin, that can remove iron from lactoferrin and

transferrin, allowing this bacterium to grow under iron-limiting conditions.

Prediction of the tertiary structure of B. pseudomallei

Partial-double-bond makes the peptide planar; it limits the rotation around C-N bond

making it to have two alpha-carbons, C, O, N and H among them in one plane. Making

thea the third angle which is omega () is constant at 1800. These three angles are the

most significant local structure parameter in protein folding. Allowed and favored regions

of dihedral angles in B. pseudomallei 1106a can be seen in figure below. Table A shows

the name, position and coordinates of residues that lie in non-core regions (outlier).

Fig. 9. Ramachandran plot of B. pseudomallei. Residues in outlier regions are numbered

accordingly.

Table 5. This table shows the name, position and coordinates of residues that lie in non-

core regions (outlier)

Name of Residue

Position of residue in sequence

Coordinates Name of Residue

Position of residue in sequence

Coordinates

Threonine (Thr)

96 -175.32, -11.24 Glycine(Gly) 454 -177.83, -58.14

Valine (V)

157 133.40, 157.46 Proline(Pro) 455 56.81, 105.40

Proline (Pro)

165 -115.66, 152.39 Arginine(Arg) 482 161.14, -43.91

Tryptophan (Trp)

173 46.95, 151.16 Lysine(Lys) 545 -65.87,-150.16

Alanine (Ala)

201 158.76,-166.87 Glycine(Gly) 549 31.09, 30.59

Aspartic acid (Asp)

236 136.98,-175.22 Proline(Pro) 568 -44.89, 163.52

Proline (Pro)

261 4.87, 40.87 Proline(Pro) 569 -43.24, -73.29

Histidine (His)

328 154.55, 128.87 Serine(Ser) 620 -7.65, -78.12

Asparagine (Asn)

345 -173.79,-140.22 Valine(V) 651 -55.41, 180.00

Threonine (Thr)

407 -65.02, -85.34 Proline(Pro) 652 6.62, 54.3

Alanine (Ala)

432 -173.28,-149.78 Arginine(Arg) 711 -159.69,-128.76

Proline (Pro)

448 -62.94,-139.77 Alanine(Ala) 735 -172.95, -69.74

Glycine has one side chain, hydrogen, while proline is limited in ramachandran plot since

phi is restricted by cyclic side chain that ranges from -35o to -85o. Clear illustrations for

glycine, pre-proline and proline residues lying in favored and allowed regions are provided

below.

Fig. 10. Ramachandran plots for glycine, preproline and proline residues. Dark colors

indicate the favored region while lighter colors designates allowed region.

Ramachandran plot illustrates the , angles of in B. pseudomallei 1106a. The

percentage of favorable, allowed and outlier region are 88.9 %, 7.5% and 3.6%,

respectively which is a little bit far from the expected value of ~98% (favorable) and ~2%

(allowed). Out of 573 residues, there are 24 residues lying in outlier regions. Outlier region

indicates how well the structures are suited in the main chain distribution of torsional

angles.

Prediction of quaternary structure

Fig. 11. Results obtained using Swiss model, given the qmean z-score, C-beta interaction

energy and AII-atom interaction energy.

Fig. 12a. 3-dimensional structure of B.

pseudomallei 1106a.

Fig. 12b. 3-dimensional structure of B.

pseudomallei 1106a based on

temperature. High values are colored in

warmer (red) colors and lower values in

colder (blue) colors.

Fig. 13. Assessment of the quality of the homology model.

QMEAN (Qualitative Model Energy Analysis) calculates global and local quality

estimates on the basis of single models. The data shown allows us to inspect the

differences between the models and helps us understand the expected accuracy of the

model.

(a) Represents the QMEAN scores of the reference structures from the PDB. It indicates

how many standard deviations the model score differs from the expected values. It has a

Z-score of -5.42 which is far from the mean. (b) This is a projection of the first plot for the

given protein size. It also shows the number of reference models used in calculation. (c)

Shows that a low quality model has a strongly negative Z-scores for QMEAN. Good

structures are said to be in the light red to blue region. The data shows that the model is

in very low quality because of its very high negative values.

Figure 14. Anolea evaluates the packing quality of the model. The Y-axis shows the

energy for each amino acid of the protein chain. Qmean estimates the global quality for

all models.

The Atomic empirical mean force potential (ANOLEA) performs energy calculations on a

protein chain. The negative energy values shown in green signify beneficial energy setting

while the positive energy values shown in red signify unfavorable energy setting for a

given amino acid. It immediately reveals if there are regions with atoms coming close to

each other and some regions have very high energy. In protein structures amino acid

residues have their preferred location. And the red regions show that the energy of the

model is much higher due to residues making bad contacts. We can conclude that the

green region is much favorable and it indicates the incorrectness of the model.

Conclusion

The species used all contained the fmtA protein; each bacterium is of different kind and

has different functions. Some are fmtA family proteins and some are ferric malleobactin

receptor and transporter. Various databases were used in comparing each species of

bacteria from the other, starting from the molecular weight, GRAVY, and number of amino

acids. Then, the matching domains and motifs are all different. Also, predicting the

location of the transmembrane helix where each has different posterior probabilities.

In doing multiple alignments, the average distance was calculated using % identity. Upon

doing that, the results came out to be B. pseudomallei that is the most distant species

among all species used.

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bioinformatics structural prediction of fmta gene from ten various species of bacteria and...

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