Evolution

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Sequence Homology

See Materials and Methods


The following query protein sequence of N-acetylneuraminic Acid was obtained from GenBank:


N-acetylneuraminic Acid
mgsdkihhhh hhmglsrvra vffdldntli dtagasrrgm levikllqsk yhykeeaeii

cdkvqvklsk ecfhpystci tdvrtshwee aiqetkggad nrklaeecyf lwkstrlqhm

iladdvkaml telrkevrll lltngdrqtq rekieacacq syfdaivigg eqkeekpaps

ifyhccdllg vqpgdcvmvg dtletdiqgg lnaglkatvw inksgrvplt sspmphymvs

svlelpallq sidckvsmsv

Fig. 1: Amino Acid Sequence (260 aa) of N-acetylneuraminic Acid (2gfh) protein from Mus musculus (House Mouse)


A total of 500 proteins were yielded. Only a 38 proteins were selected for comparison as these had shown higher homology to the query sequence, in contrast with the remainder of the search results.


These proteins were chosen according to their bit scores and E-values. Two more outlier partial sequences contributing to poor overall alignment (huge deletion gaps) were subsequently removed. The remaining 36 sequences were used for the generation of the phylogenetic tree (and bootstrapped tree as well).


The bacteria sequence matches used for multiple sequence analysis and phylogenetic tree mapping were representative of the other baterial sequences not selected. These selected sequences had the highest bit scores and E-values.


Multiple Sequence Alignments (msa)

See Materials and Methods


The following sequence alignment was obtained (Fig. 2)


From the alignments, gi|10888xy and gi|10888yz are representative of gi|108881764 and gi|108881765 respectively. Both these hypothetical proteins belong to the mosquito Aedes aegypti.


The identifier numbers for these two proteins were initially changed to an alpha-numeric one, due to the inability of Phylip to generate a tree from the original identifiers. This was due to the fact that the programme only took the first five numeric digits (10888), thereby resulting in a programme error prompt which listed both proteins as duplicates (from the identifier numbers). Both these identifiers were subsequently renamed for the final phylogenetic tree.


The topmost sequence belongs to the query protein.


msa


msa


msa

Figure 1. Multiple Sequence Alignments of 2gfh amino acid sequences with that of other similar protein sequences in various organisms.


From the multiple sequence alignment, it can be observed that there are domain conservations throughout the protein sequences. Small insertion and deletion gaps were noticeable along the alignment as well. A particularly large insertion gap was observed between amino acids 91 to 114.


The organisms with the large insertion gaps were as identified below:


  1. Bacillus licheniformis
  2. Bacillus subtilis
  3. Bacillus halodurans
  4. Bacillus clausii
  5. Symbiobacterium thermophilum


All five sequences belong to bacteria. With the exception of Symbiobacterium thermophilum, the rest are Bacillus sp.


MediaWiki


Symbiobacterium is an uncultivable thermophile isolated from compost. Its survival is based mainly on microbial commensalisms (1). This bacteria can only grow in vitro, if it is co-cultured with Bacillus sp. bacteria (1). This could therefore explain its genetic association with Bacillus, as observed from the sequence alignment.

However, interestingly, Bacillus is classified as Gram-positive, while Symbiobacterium is a Gram-negative bacterium. As observed from the sequence alignment, other Gram-negative bacterium protein sequences (Vibrio Sp.) do not contain the large gap insertion at the 91 to 114 amino acid positions, with the exception of Symbiobacterium. Hence, more genetic (and even functional) analysis might be necessary to determine the hydrolase protein relationship between the Gram-positive Bacillus with the Gram-negative Symbiobacterium.


A highly conserved (with invariant) section of amino acids (LV)–(LVA)–(LIV)–(LIV)-T-N-G was observed in all the sequences from amino acid 211 to 217 in the alignment. Downstream of this conserved portion of genes are 5 more invariant positions (1 or 2 amino acids in length).


From these short conservation regions, the functions or even structure of the encoded proteins could have significance in its evolutionary pattern.

Phylogenetic Map (Tree)

See Materials and Methods


The tree was plotted to obtain the phylogenetic lineage (Fig. 3):


Radial View




Cladogram View


Fig. 3: Phylogenetic tree showing organisms with related protein sequence homology in Radial Tree view (top); and Rectangular Cladogram view (bottom)


From the Rectangular Cladogram view, it could be observed that there are four distinct separate groups.


Bootstrapping

See Materials and Methods


Bootstrapping values obtained were analysed. Branch values occurring below 75% (<75%) are indicated by an asterisk (*) [Fig. 4].


Bootstrap Values

Fig. 4: Branch bootstrap values in Rectangular Cladogram view. Branches with strap values <75% were indicated with asterisks (*)



Table of functions
Catalytic activity N-acylneuraminate 9-phosphate + H2O = N-acylneuraminate + phosphate
Cofactor Magnesium (By similarity)
Enzyme regulation Inhibited by vanadate and calcium (By similarity)
Pathway Carbohydrate metabolism; aminosugar metabolism
Similarity Belongs to the haloacid dehalogenase-like hydrolase superfamily. NANP family



Separate Enzymes Catalyze the Final Two Steps of Coenzyme A Biosynthesis in Brevibacterium ammoniagenes: Purification of Pantetheine Phosphate Adenylyltransferase