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Evolution, Structure and Function of N-acetylneuraminic Acid Phosphatase


Jason Cheong Wen Leong (s41235935), Yau Heen wai (s41286272), Lim Junxian (s41313011)


Abstract


N-acetylneuraminic acid phosphatase a novel protein investigated by our group. With its structure and sequence known, the function was

assumed to be a part of the enormous family of haloacid dehalogenase-like hydrolases. It represent the family of predicted small molecule

phosphatases related by sequence cleave sites and reactions in the genomes of bacteria, archaea, and eukaryotes. Many have evolved to be used

for specific biological functions within individual organism


Introduction


The novel protein investigated by our group is N-acetylneuraminic acid (Neu5Ac) phosphatase, it was first release on Protein Data Bank

(PDB) on 18th April 2006, named 2gfh. Mus muscular (mouse) was used as the source of the gene and Escherichia coli was the

vector used to express the novel protein. In Homo sapiens (man), it was known to be as N-acetylneuraminate 9-phosphate (Neu5Ac-9-P)

phosphatase haloacid dehalogenase (HAD)-like hydrolase domain containing protein 4. Other aliases of the novel protein include C20orf147, NANP

and HDHD4. The gene encoding the protein was found to be on chromosome 20; location 20p11.1.


Neu5Ac-9-P phosphatase belongs to a large family of haloacid dehalogenase (HAD)-like hydrolases. The enzymes found within this classification

possess varied types of cleavage activities. Although many of its members are related by sequence cleave sites and reactions, many have evolved

to be used for specific biological functions within individual organisms.


These small molecule phosphatase enzymes have been found to exists in the various domains of life — Bacteria, Archaea, and Eucarya. The number

of genes found within each organism is varied from bacteria to eukaryotes. Bacterial Neu5Ac synthase and mammalian Neu5Ac-9-P synthase are

homologous proteins, sharing about 35% sequence identity1. Neu5Ac-9-P phosphatase dephosphorylates Neu5Ac-9-P to form Neu5Ac, the

main form of sialic acid.

Document2 01.png

Figure 1. Dephosphorylation of Neu5Ac-9-P is a reversible reaction with an end product of Neu5Ac (sialic acid) and a free phosphate.


Sialic acids are nine-carbon sugars with a carboxylate group that are found as components of many glycoproteins, glycolipids, and

polysaccharides in animals, viruses, and bacteria. The main form of sialic acid, Neu5Ac, is often present as the terminal sugar of N-

glycans on glycoproteins and glycolipids and plays an important role in protein–protein and cell–cell recognition 2; 3.

Document5 01.png

Figure 2. Chemical structure of sialic acid.(http://en.wikipedia.org/wiki/Sialic_acid)


Sialic acids are found widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. The amino group

bears either an acetyl or a glycolyl group. Sialic acid consists of acetylated, sulfated, methylated, and lactylated derivatives and is a large

family of more than 50 members 4.


Results


Query Sequence


The amino acid query sequence of 2gfh protein (Figure 3) from Mus musculus is obtained from Genbank.


<1 mgsdkihhhh hhmglsrvra vffdldntli dtagasrrgm levikllqsk yhykeeaeii

61 cdkvqvklsk ecfhpystci tdvrtshwee aiqetkggad nrklaeecyf lwkstrlqhm

121 iladdvkaml telrkevrll lltngdrqtq rekieacacq syfdaivigg eqkeekpaps

181 ifyhccdllg vqpgdcvmvg dtletdiqgg lnaglkatvw inksgrvplt sspmphymvs

241 svlelpallq sidckvsmsv>

Figure 3. The 260 amino acid sequence of 2gfh protein.


Sequence Homology


From the BlastP similarity was used for comparison as these had shown higher homology to the query sequence sequence search, a total of 500

proteins were yielded.Only a total of 38 proteins, 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).


Multiple Sequence Alignment


The following multiple sequence alignment (MSA) was obtained (Figure 4). 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.


Document2 04.png

Figure 4. MSA of query (top-most sequence – No.1) and related sequences.


From the MSA, it can be observed that there are generally slight 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:

Bacillus licheniformis

Bacillus subtilis

Bacillus halodurans

Bacillus clausii

Symbiobacterium thermophilum


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 Tree


The tree was plotted to obtain the phylogenetic lineage (Figure 5).


Document6 01.png
Document6 02.png


Figure 5. (A) Phylogenetic tree showing organisms with related protein sequence homology in Radial Tree view. (B) Rectangular

Cladogram view with related protein sequence homology.


From the Rectangular Cladogram view, it could be observed that there are four distinct separate groups involving fishes, mammals (where the

query protein is also mapped), bacteria and insects.


Bootstrapping


Bootstrapping values obtained were analysed. Branch values occurring below 75% (<75%) would be indicated by an asterisk (*),

as shown in Figure 6.

Document7 01.png

Figure 6. Branch bootstrap values in Rectangular Cladogram view. Branches with strap values <75% were indicated with

asterisks (*)


DALI Searching

SUMMARY: PDB/chain identifiers and structural alignment statistics NR. STRID1 STRID2 Z RMSD LALI LSEQ2 %IDE REVERS PERMUT NFRAG TOPO PROTEIN

 1: 3033-A 2gfh-A 41.1  0.0  246   246  100      0      0     1 S    HYDROLASE        haloacid dehalogenase-like hydrolase domain
 2: 3033-A 1fez-A 18.1  3.5  178   256   22      0      0    13 S    HYDROLASE        phosphonoacetaldehyde hydrolase         (bacillus c 
 3: 3033-A 2hsz-A 17.9  3.3  168   222   23      0      0    13 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    novel predicted
 4: 3033-A 1qq5-A 17.3  3.1  198   245   19      0      0    12 S    HYDROLASE        l-2-haloacid dehalogenase       (xanthobacter aut 
 5: 3033-A 1o03-A 17.0  5.0  188   221   20      0      0    11 S    ISOMERASE        beta-phosphoglucomutase         (lactococcus lactis
 6: 3033-A 2b0c-A 16.4  2.6  184   199   20      0      0    13 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    putative phospha 
 7: 3033-A 2fdr-A 15.8  4.4  190   214   19      0      0    15 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    conserved hypoth
 8: 3033-A 2p11-A 15.7  2.9  194   211   16      0      0    20 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro 
 9: 3033-A 1te2-A 15.7  3.6  170   211   19      0      0    15 S    HYDROLASE        putative phosphatase    (escherichia coli o157
10: 3033-A 1yns-A 15.3  4.0  169   254   11      0      0    13 S    HYDROLASE        e-1 enzyme (enolase-phosphatase e1)     (homo s 
11: 3033-A 1qyi-A 15.0  3.5  198   375   19      0      0    17 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro
12: 3033-A 2i6x-A 14.9  3.1  176   199   19      0      0    18 S    HYDROLASE        hydrolase, haloacid dehalogenase-like family 
13: 3033-A 1u7p-A 14.3  2.9  144   164   18      0      0    14 S    HYDROLASE        magnesium-dependent phosphatase-1 (mdp-1)       (
14: 3033-A 1ymq-A 14.1  2.3  130   260   16      0      0    14 S    TRANSFERASE      sugar-phosphate phosphatase bt4131      (bacte 
15: 3033-A 1j8d-A 13.1  2.5  141   180   11      0      0    12 S     HYDROLASE       deoxy-d-mannose-octulosonate 8-phosphate ph
16: 3033-A 2ho4-A 12.9  2.4  131   246   19      0      0    14 S    HYDROLASE        haloacid dehalogenase-like hydrolase domain 
17: 3033-A 1pw5-A 12.7  2.3  136   246   21      0      0    12 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    nagd protein, pu
18: 3033-A 1nf2-A 12.7  2.6  127   267   13      0      0    11 S    STRUCTURAL GENOMICS/UNKNOWN FUNCTION     phosphatase     (the 
19: 3033-A 1rlm-A 12.4  2.8  131   269   13      0      0    14 S    HYDROLASE        phosphatase Mutant      (escherichia coli) bacte
20: 3033-A 1f5s-A 12.1  3.5  159   210   14      0      0    15 S     HYDROLASE       phosphoserine phosphatase (psp)         (methanoco 
21: 3033-A 1cr6-B 12.0  3.8  177   541   18      0      0    18 S    HYDROLASE        epoxide hydrolase       (mus musculus) mouse expr
22: 3033-A 1rku-A 11.9  3.6  172   206   11      0      0    18 S    TRANSFERASE      homoserine kinase       (pseudomonas aeruginosa 
23: 3033-A 2b30-A 11.8  2.7  134   284   16      0      0    12 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    pvivax hypotheti
24: 3033-A 1kyt-A 10.5  2.5  122   216   13      0      0    15 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro 
25: 3033-A 2o2x-A 10.3  3.6  139   204   17      0      0    14 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro
26: 3033-A 1u02-A 10.1  2.7  128   222   16      0      0    12 S    STRUCTURAL GENOMICS      trehalose-6-phosphate phosphatase 
27: 3033-A 2fea-A 10.0  3.5  167   219    7      0      0    21 S    HYDROLASE        2-hydroxy-3-keto-5-methylthiopentenyl-1- pho
28: 3033-A 2hx1-A  9.6  3.2  130   275   24      0      0    19 S    HYDROLASE        predicted sugar phosphatases of the had supe 
29: 3033-A 1mh9-A  9.2  3.2  146   194   15      0      0    15 S    HYDROLASE        deoxyribonucleotidase (mitochondrial 5'(3')-

Figure 7. The DALI search results that were returned through e-mailed. The highlighted (yellow) shows the query protein. With a z value

of 41.1 and a root mean standard deviation of 0.0 and %IDE of 100, shows that it is a HAD family protein. The highlighted (green) shows

significant similarities of query protein as a hydrolase phosphatase as Z values are more then 1, RMSD still of low values and %IDE of more

then 20.Z


From the DALI search (Figure 7), Neu5Ac phosphatase is a haloacid dehalogenase-like hydrolase. This family is structurally different from the

alpha/ beta hydrolase family. It has L-2-haloacid dehalogenase, epoxide hydrolases and phosphatases. This family consists of two domains of

structure. One is an inserted four helix bundle, which is the least well conserved region of the alignment, between residues 16 and 96 of (S)-2-

haloacid dehalogenase I. The remaining of the fold is composed of the core alpha/beta domain. It is classified as a hydrolase found in mouse.

The chemical components would be phosphate ion, sodium ion, 1,2-ethanediol, chloride ion. PO4 and EDO are ligands while

Na and Cl are metals.