CHAPTER 8 COMPARISON OF NTN -HYDROLASES INCLUDING NTN ... 8.pdf · COMPARISON OF NTN -HYDROLASES INCLUDING…

Download CHAPTER 8 COMPARISON OF NTN -HYDROLASES INCLUDING NTN ... 8.pdf · COMPARISON OF NTN -HYDROLASES INCLUDING…

Post on 04-Jun-2018

215 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • 239

    CHAPTER 8

    COMPARISON OF NTN-HYDROLASES

    INCLUDING NTN-HYDROLASE DOMAINS

    8.1 Introduction

    To compare the Ntn-hydrolase superfamily of proteins we have divided them into

    three categories based on the type of N-terminal nucleophile residue, which is a cysteine,

    serine or a threonine. An extensive sequence comparison and analysis was carried out in

    each category separately. Many related proteins from eukaryotes in the database were

    identified in serine and cysteine groups. In the category where threonine was the N-

    terminal nucleophile residue two distinct groups could be identified based on the

    closeness of amino acid sequences. Thus, through careful sequence comparison we not

    only could identify new, but distantly related, Ntn-hydrolase members or domains but

    also could place in this family some of the un-annotated proteins in the database.

    A variety of enzymes with varied substrate specificity, classified by their

    characteristic and distinct fold, form the N-terminal nucleophile (Ntn) hydrolase

    superfamily. Despite lack of any discernible sequence similarity, the representative

    structures of Ntn hydrolases show that similar fold and topological coincidence spatially

    conserve the amino acid residues important for activity. Because of the spatially

    conserved active site they are also mechanistically related. However, the nature of the

    nucleophile residue, oxyanion hole residues and topology of binding sites greatly differ.

    The evolution of enzyme function and the nuances of catalysis of Ntn hydrolases can be

    fully deciphered only by a complete analysis of the sequences and structures along with

    corresponding detailed phylogenetic analysis. The structural analysis of individual

    members of superfamily has revealed how nature has optimized binding and catalysis,

    and re-structured old proteins for new activities through gene duplication and mutation

    (Kumar et al., 2006).

  • 240

    Statistics of Ntn-hydrolase family (adopted from phylofacts database - http://phylogenomics.berkeley.edu/)

    Superfamily code : 56235 Fold name : Ntn hydrolase -like No of genomes : 275 No of Phyla : 22 No of sequences : 867 Average size : 236 Diversity : 0.132897 In every individual of the family the terminal of one of the -strands of the

    characteristic fold is decorated with the nucleophile residue, a Ser, Cys or Thr

    whose free -amino group act as the base in catalysis (Brannigan et al., 1995). Minor

    modification of the oxyanion hole occurs in terms of the residues involved depending

    also on the type of nucleophile residue present at the N-terminus. Based on the N-

    terminal nucleophile residue these hydrolases can be widely classified into three sub-

    groups/families, of those possessing a cysteine, serine, or a threonine at the N-terminus.

    Well-refined representative structures for all three types, the Cys-, Ser- and Thr-families

    exist. Here we have used the representative sequences and structures of PVA and BSH

    for Cys-family, that of PGA for Ser-family and that of L-asparaginase (Flavobacterium

    Meningosepticum) for Thr-family. The presence of Ntn-hydrolases span over several

    organisms, both prokaryotes and eukaryotes. They exist as single functional protein

    molecule or as part of a protein domain. The Pei & Grishin (2003) has identified that U34

    peptidase family belonged to the Ntn hydrolase fold and consisted of choloyglycine

    hydrolases, acid ceramidases, isopenicillin N acyltransferases, and a subgroup of proteins

    with unclear function. A multiple sequence alignment arranges the protein sequences into

    a rectangular array so that residues in a given column are homologous, superposable or

    plays a common functional role (Edger & Batzoglou, 2006). Based on their amino acid

    sequences and structural information, attempt is made here to organize these proteins

    phylogenetically and functionally into sub-families depending on their sequence-

    relationship, substrate specificities and evolutionary closeness.

    In the study reported here extensive sequence analysis is carried out to identify

    different protein families belonging to Ntn-hydrolase superfamily and to understand their

    functional and evolutionary relationships.

  • 241

    8.2. Results

    8.2.1. Penicillin V acylase: N-terminal cysteine nucleophile (Ntcn) hydrolase

    Peptidases are a diverse group of enzymes that hydrolyse the peptide bonds in

    protein, peptides and various other molecules. These peptidases are classified based on

    the participating residues in the catalysis. The new family of Ntn hydrolases, although

    similar to peptidases in terms of the type of bonds they cleave, they are identified more as

    amidases and they show great economy in terms of those groups participating in the

    catalytic activity. In contrast to common peptidases in which catalytic center is made up

    of a triad of three groups, Ntn hydrolase are made up of a single catalytic center. A base

    adjacent to the catalytic amino acid is necessary and expected to enhance the nuclophilic

    character of the side chain nucleophile groups (-OH or -SH). Very often there is a

    bridging water molecule from nucleophile atom to the free -amino group in the same

    residue which act as base. Some of the peptidases like U34 family are recently identified

    to belong to Ntn hydrolase superfamily using extensive sequence analysis and the fold

    characteristics (Pei & Grishin., 2003). The members of this family exhibit considerable

    sequence variation and individuals show wide specificity towards a variety of substrates.

    Using the sequence of BspPVA as query a protein-protein Blast search was

    conducted with default input parameters which output many protein sequences of PVA

    and BSH from diverse sources, mainly from microorganisms. To identify homologous

    proteins in higher organisms analysis of a group classified as cholylglycine hydrolases in

    Pfam (Batman et al., 2002) was carried out. It has now established that bile salt hydrolase

    is very closely related to PVA, evident from the similarity in active site residues and

    substrate recognition and binding (Kumar et al., 2006). The three-dimensional structures

    are also exceptionally similar with differences mainly confine to substrate binding loop

    that play role in substrate specificity. A sequence homology analysis and structural

    comparison of BSH and PVA revealed that four of the five amino acids at the active site

    of PVA are conserved in BSH (Tanaka et al., 2001). Although sequence and structure of

    PVA and BSH are very similar, differences are observed in certain critical positions.

    Further investigations are necessary to explore the role of residues in these key positions

    responsible for substrate selectivity

  • 242

    Figure 8.1: sequence alignment of BSH with ASAH of human and mouse. Arrows

    indicate the positions of conservation of crucial amino acids between BSH

    and ASAH.

    Choloylglycine hydrolase family in Pfam database contains 132 homologous

    sequences from different organisms. The N-acylsphingosine amidohydrolase (ASAH)

    also called as Putative 32 kDa heart protein, sequence from mouse was selected and

    protein-protein Blast was repeated again. The Blast gave 68 hit sequences. Sequence

    alignment was performed using sequences obtained from Blast using BlBSH as reference

    sequence and ASAH protein from mouse used as query sequence. In humans, the N-

    acylethanolamine-hydrolyzing acid amidase that hydrolyse various N-acylethanolamines

    has N-palmitylethanol-amines as the most reactive substrates. And they are identical to

    acid ceramidase but lack ceramide hydrolyzing activity (Hassler and Bell, 1993). The

    sticking sequence similarity between BlBSH and Human ASAH and ceramidase are

    clearly depicted in figure 8.1.

    Figure 8.1: sequence alignment of BSH with ASAH of human and mouse. Arrows

    indicate the positions of conservation of crucial amino acids between BSH

    and ASAH.

    Choloylglycine hydrolase family in Pfam database contains 132 homologous

    sequences from different organisms. The N-acylsphingosine amidohydrolase (ASAH)

    also called as Putative 32 kDa heart protein, sequence from mouse was selected and

    protein-protein Blast was repeated again. The Blast gave 68 hit sequences. Sequence

    alignment was performed using sequences obtained from Blast using BlBSH as reference

    sequence and ASAH protein from mouse used as query sequence. In humans, the N-

    acylethanolamine-hydrolyzing acid amidase that hydrolyse various N-acylethanolamines

    has N-palmitylethanol-amines as the most reactive substrates. And they are identical to

    acid ceramidase but lack ceramide hydrolyzing activity (Hassler and Bell, 1993). The

    sticking sequence similarity between BlBSH and Human ASAH and ceramidase are

    clearly depicted in figure 8.1.

  • 243

    Choloylglycine hydrolase & PVA from Bacillus (cereus, thuringiensis,

    anthracis)

    Bile salt hydrolases

    N-acylsphingosine amidohydrolase (even from Homo sapiens)

    N-acylethanolamine-

    hydrolyzing acid amidase

    Hypothetical and unnamed protein products

    PVA & related proteins, Choloylglycine hydrolase

    Bacillus Sphaericus

    Bacillu