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Research Article
Molecular Phylogeny and Predicted 3D Structure of
Plant beta-D-𝑁-Acetylhexosaminidase
Md. Anowar Hossain1,2 and Hairul Azman Roslan2
1Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi 6205, Bangladesh
2Genetic Engineering Laboratory, Department of Molecular Biology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Correspondence should be addressed to Md. Anowar Hossain; mahossain@hotmail.com
Received February ; Revised June ; Accepted June ; Published July
Academic Editor: Xiu-feng Wan
Copyright © Md. A. Hossain and H. A. Roslan. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
beta-D-𝑁-Acetylhexosaminidase, a family glycosyl hydrolase, catalyzes the removal of 𝛽-,-linked 𝑁-acetylhexosamine
residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of 𝛽-hexosaminidases to analyze the
evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history
of evolution of plant 𝛽-hexosaminidase that can be described by gene duplication events. e D structure of tomato 𝛽-
hexosaminidase (𝛽-Hex-Sl) was predicted by homology modeling using now as a template. Structural conformity studies of the
best t model showed that more than % of the residues lie inside the favoured and allowed regions where only .% lie in the
unfavourable region. Predicted D structure contains amino acids residues with glycosyl hydrolaseb domain-I and glycosyl
hydrolase superfamily domain-II including the (𝛽/𝛼)8barrel in the central part. e 𝛼and 𝛽contents of the modeled structure
were found to be .% and .%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp() and
Glu() could play important roles in enzyme-catalyzed reactions. e predicted model provides a structural framework that can
act as a guide to develop a hypothesis for 𝛽-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in
plant kingdom.
1. Introduction
Asapartofthestudytoelucidatetheroleoffree𝑁-glycans
and de-𝑁-glycosylation mechanism working in plants,
we have already characterized the PNGase, ENGase, 𝛼-
mannosidase and 𝛽-hexosaminidase at molecular level [–].
e 𝛽-D-𝑁-acetylhexosaminidase (EC ...), a member
of the glycosyl hydrolase family (GH), is an enzyme that
hydrolyses nonreducing terminal monosaccharide residues
of 𝛽-𝑁-acetylgalactosaminides and 𝛽-𝑁-acetylglucosami-
nides. It is widely distributed among the animals, insects,
plants, fungus, and bacteria. Mammal lysosomal 𝛽-𝑁-acetyl-
D hexosaminidases are mainly responsible for glycoconjugate
degradation in lysosome. HexA is a heterodimer of subunits
𝛼(encoded by the gene HexA) and 𝛽(encoded by the
gene HexB), whereas HexB is a homodimer of 𝛽subunits.
e subunits arose through a gene duplication event and
the primary sequences are approximately % identical.
Mutational defects that cause 𝛽-hexosaminidase-A and B
deciency are responsible for Sandho and the Tay-Sachs
diseases, respectively []. Recently, it has been reported that
𝛽-hexosaminidase is a surrogate marker for renal function in
autosomal dominant polycystic kidney disease []. In insects,
ithasbeenpostulatedtohavespecializedphysiological
functions, including posttranslational modication of
𝑁-glycans, degradation of glycoconjugates, and egg-
sperm recognition, suggesting that these enzymes have
rather versatile physiological functions in the growth
and development of insects []. Mammal 𝛽-𝑁-acetyl-D-
hexosaminidaseshavebeenshowntobeimportantfor
egg-sperm recognition [], and the enzymes from Drosophila
melanogaster sperm membrane also participate in the
same process []. A fungal 𝛽-𝑁-acetyl-D-hexosaminidases
has been expressed, characterized, and crystallized from
Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 186029, 14 pages
http://dx.doi.org/10.1155/2014/186029
e Scientic World Journal
Aspergillus oryzae, which has sequence similarity to bacterial
and human enzymes ranges from % to % [].
Recently, plant 𝛽-𝑁-acetyl-D-hexosaminidases has
gained a lot of attention due to its presence in the ripening
stages []. It has also been shown that the tomato fruit
shelflifecanbeenhancedbythesuppressionof𝑁-glycan
processing/degrading enzymes []. Plant glycoproteins
contain substantial amounts of paucimannosidic 𝑁-glycans
lacking terminal GlcNAc residues at their nonreducing
ends. It has been proposed that this is due to the action of
𝛽-hexosaminidases during late stages of 𝑁-glycan processing
orinthecourseof𝑁-glycan turnover []. Although several
𝛽-hexosaminidases have been reported from various parts
of plants such as leaves, fruits, and seeds [–], their
physiological functions in plant biology are not yet fully
understood. To elucidate the exact roles of this enzyme
in plant kingdom, it is desirable to know about properties
and behavior of the phylogenetically related enzymes from
dierent species and their molecular evolutions. However,
little is known about the phylogenetics and evolution of plant
𝛽-hexosaminidases.
So far eight crystal structures of GH 𝛽-𝑁-acetyl-D-
hexosaminidases have been reported including two humans,
one insect, and six bacterial enzymes. Both the human
HexA and HexB are the 𝛽-𝑁-acetyl-D-hexosaminidases that
degrade glycoconjugate in the lysosome [,]. OfHex,
theenzymefromtheAsiancornborerOstrinia furnacalis
(one of the most destructive pests), has been reported to
function merely in chitin degradation []. e bacterial
enzymes include SpHex and SmCHB, which are found in
the chitinolytic bacteria Streptomyces plicatus and Serratia
marcescens,respectively[–]. AaDspB, which is isolated
from Aggregatibacter actinomycetemcomitans,isinvolvedin
the degradation of biolm (polymeric 𝛽-,-linked GlcNAc)
[]. e enzyme, PsHex from Paenibacillus sp.TS,can
eciently degrade various glycosphingolipids []. PgGcnA,
theenzymefoundintheendocarditispathogen,Strepto-
coccus gordonii,isinvolvedinthereleaseofdietarycar-
bohydrates []. Recently, it has been found that a novel
𝛽-𝑁-acetylhexosaminidase, StrH protein from Streptococcus
pneumoniae R6, is involved in the catalytic specicity towards
the 𝛽(,)-linked 𝛽-𝑁-acetylglucosides and key residues
intheactivesiteareTrp-andTyr-[]. us, it
is interesting to know how these enzymes could carry
out their specialized functions in terms of their structural
features. To our knowledge, no crystal structure of plant 𝛽-𝑁-
acetyl-D-hexosaminidase has yet been reported. erefore,
comparative homology modeling of tomato 𝛽-𝑁-acetyl-
D-hexosaminidase is desirable to elucidate the functional
prediction, active site information, and mechanism of action.
Inthepresentwork,rstweidentiedthehomologous
sequences of 𝛽-𝑁-acetyl-D-hexosaminidase in GenBank by
theNCBIBLAST-PSIsearch.Wedidmultiplesequencealign-
ments and reconstructed the phylogenetic tree. Secondly,
in order to initiate structural studies of this enzyme, we
performed sequence alignment and D-structure homology
modeling and constructed a molecular model of this enzyme
and of its complex with the natural substrate. We also
performed molecular docking of the enzymes and predicted
theactivesiteresiduesresponsibleforcatalyticactivity.
e predicted D structural information will be useful to
study the site-directed mutagenesis wet lab experiments as
well as the physiological functions of tomato 𝛽-𝑁-acetyl-D-
hexosaminidase in the plant kingdom.
2. Material and Methods
2.1. Data Retrieval. In this study, we retrieved all of the
sequences from the National Center for Biotechnology
Information (NCBI) GenBank database as described by
Gonzalez and Jordan []. Shortly, an initial dataset of
the previously published and functionally characterized
𝛽-𝑁-acetyl-D-hexosaminidase amino acid sequences was
retrieved manually from Entrez (http://www.ncbi.nlm.nih
.gov/entrez). e representative sequences including the
𝛽-Hex-Sl were isolated from a wide phylogenetic range of
eukaryotes and prokaryotes, which possessed a variety of bio-
chemical activities. A CD-Hit clustering program was used to
group these sequences by amino acid identities into clusters
[]. Divergent 𝛽-𝑁-acetyl-D-hexosaminidase amino acid
sequences with representatives from each cluster were
used as queries in a series of PSI-BLAST (Position-Specic
Iterated BLAST) searches of the protein database throughout
all organisms at NCBI []. e representative sequences
were Solanum lycopersicum beta-hexosaminidase sequence
[gi:], Arabidopsis thaliana AtHex[gi:],
Homo sapiens protein sequences HexA[gi:]and
HexB[gi:],Drosophilamelanogasterhexosaminidase
sequences Hexo[gi:]and Hexo[gi:],
Aspergillus oryzae HexA[gi:], and Streptomyces
plicatus HexA[gi:]. We chose the sequences from
BLAST results based on the high similarities of amino acids
(>% identities) with the query representative sequences.
e picked sequences were checked manually to exclude
incomplete and redundant sequences. For the feature analysis
and construction of phylogenetic tree we took a total of
sequences, which are already characterized as predicted or
true 𝛽-𝑁-acetyl-D-hexosaminidase from the GenBank, to
reduce computational burden. An archea sequence was also
retrieved from GenBank that was used as an outgroup in the
construction of phylogenetic tree.
2.2. Multiple Sequence Alignments and Construction of Phy-
logenetic Tree. MUSCLE program []wasusedtoalignall
amino acid sequences of 𝛽-𝑁-acetyl-D-hexosaminidases
and the alignments were checked manually. Unambiguously
aligned regions were identied using GBlocks program [].
e phylogenetic relationships between the genes were ana-
lyzed using the maximum-likelihood (ML) method. For the
ML analyses, we used the PROTML program of PHYLIP
version . [].
We employed the WAG model of amino acid substitution
withgammadistributionsiterateandinvariablesitecategory
for phylogenetic analysis []. All indels were counted as
missing. We performed ten random sequence addition
searches using the J option and global branch swapping
using the G option to isolate the ML tree with the best log
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Active site
Query seq.
1 100 200 300 400 500 575
GH20 hexosaminidase superfamily
GH20 HexA HexB-like
Glyco hydro 20
Glyco hydro 20b
Specic hits
Multidomains
Superfamilies
F : Conserved domains for tomato 𝛽-hexosaminidase, analyzed using Conserved Domain Database search in NCBI-BLAST.
likelihood. In addition, we performed bootstrap analysis
with replications.
2.3. Comparative Homology Modeling of Tomato 𝛽-𝑁-Acetyl-
D-hexosaminidase. AminoacidsequenceofSolanum lycop-
ersicum 𝛽-𝑁-acetyl-D-hexosaminidase (𝛽-hex-Sl) composed
of residues was retrieved from NCBI GenBank (GI:
and Accession no. NP .). e SWISS-
MODEL web server [] was used to identify the template
structure, now, and also used for homology modeling.
e online ModWeb Comparative Modeling Server version
SVN.r:M and I-TASSER []werealsousedfor
further modeling to compare which is the most correct
model. e DFire [], QMEAN [], PROCHECK [],
WHAT CHECK [], and VERIFY D []methodsand
ModEval model evaluation server []wereusedtocheck
the validity of the modeled structures. UCSF Chimera and
Swiss-PdbViewer were used to view the models and images
preparation. e COFACTOR, a structure-based method for
biological function annotation of protein molecules, was used
to identify the functional insights including ligand-binding
site, gene-ontology terms, and enzyme classication [–].
3. Results and Discussion
3.1. Sequence Analysis of 𝛽-Hex-Sl. e 𝛽-Hex-Sl protein
sequence was analyzed by NCBI CD-search tool (CDD
V.- PSSMs) to identify the conserved domains
(CD).esequencecontainsaGlyco hydro b (∼ aa),
GH HexA HexB-like domain (∼ aa), and a glycosyl
hydrolase family , catalytic domain (∼ aa) belongs to
the GH hexosaminidase superfamily proteins (Figure ).
Based on CD database available and three-dimensional
structure-activity relationship, the amino acid residues
Arg(), Asp(), His(), Asp(), Glu(), Trp(),
Trp(), Tyr(), Asp(), Trp(), and Glu() were
predicted to be present in the active site of 𝛽-Hex-Sl with
other sequences (Figure ). e online tool NetNGlyc .
server was used to identify the 𝑁-glycosylation site present
intheproteinsequence.epredicted𝑁-glycosylation sites
were position at (NFTI), (NLTS), (NESY),
(NPTR), (NPSI), (NGTL), (NNTL), (NRTV),
(NPSL), (NNTK), and (NDSR) (data not shown).
e soware SignalP . server was used to predict the
signalpeptidecleavagesitethatwasfoundtobeinbetween
positions and in the amino acid sequence.
3.2. Phylogenetic Analysis of 𝛽-Hexosaminidase Sequences.
In order to know the evolutional history and properties
of plant beta-hexosaminidases, we reconstructed the phy-
logenetic tree. We aimed to collect the sequence data of
the beta-hexosaminidases from a wide range of organisms
so that we could get a lot of information including their
physicochemical, structural, and biological functions. A total
of amino acid sequences were retrieved from the Gen-
Bank database by previously characterized representative
sequences. ese sequences used in the analysis include
experimentally characterized 𝛽-𝑁-acetyl-D-hexosaminidase
enzymesaswellasnovelpredictedorputative𝛽-𝑁-acetyl-
D-hexosaminidase sequences (Table ). MUSCLE program
was also used to align the sequences, whereas maximum
likelihood method was used in phylogenetic reconstruc-
tion. Our phylogenetic analysis shows that 𝛽-𝑁-acetyl-D-
hexosaminidases are widely distributed among plant, animal,
insects, fungi, and bacteria, belonging to the glycosyl hydro-
lase superfamily (Figure ). It reveals the complex history
of evolution of 𝛽-𝑁-acetyl-D-hexosaminidases that can be
described by multiple gene duplication events.
Eukaryotic 𝛽-hexosaminidases might be originated from
common bacterial ancestor through multiple gene duplica-
tions. Bacteria and fungi clades mostly contain one gene for
hexosaminidase in each species albeit few have two genes.
Bacteria clade consists of 𝛽-hexosaminidases that have the
peptidoglycan degradation and chitinolytic activities. ose
bacterial species, which contain two genes of hexaminidases,
mightacquiretheirlastcopieseitherbyhorizontalgenetrans-
fer or gene duplication. Fungi sequences clearly showed its
owncladeandonlyfewspecieshavemorethanonegeneand
might be originated either lineage specic mutation and/or
gene duplication. Insects clade-I and clade II and plants clade-
I and II also contain at least one hexosaminidase gene in each
species. Insects (I and II) clades hexosaminidases are chiti-
nolytic enzymes, which separately form paraphylactic groups
that could be evolved by gene duplication. Plants clade-I and
clade-II also constitute paraphylactic group and also split
into monocotyledons and dicotyledons that have functional
divergences. Plant 𝛽-hexosaminidases are involved in 𝑁-
glycan processing of cell walls. Animal clade clearly splits into
two clades, A and B, that contain the isoenzymes, HexA and
HexB, respectively.
Gene duplication is considered a major driving force for
evolution of genetic novelty, thereby facilitating functional
divergence and organismal diversity, including the process
of speciation. It can be generated by several mechanisms,
including tandem duplication, transposition, and large-
scale duplication (e.g., segmental/whole genome duplication
(WGD)). Also, segmental duplications (SDs) are increasingly
recognized as frequent phenomena, especially in primate
genomes; for example, approximately % of the human
e Scientic World Journal
Feature 1
1NP0_B 151 FSH RGILI DTSRHYLPVKIILKTLDA MAFNKFNVL HWH IVDDQ SFP YQSITF P.[12].VYTPN DVRMVIEYARLR GIRV 236
query 167 FTH RGVML DTS RNFYGVDHLLRLIKA MSMNKLNVFHWH ITDSHSFP LVIPSE P.[12].MYSPA DVQKIVEYGMEH GVRV 252
gi 24653074 276 FRY RGLML DTSRHFFSVESIKRTIVG MGLAKMNRF HWH LTDAQ SFP YISRYY P.[12].TYSEQ DVREVAEFAKIY GVQV 361
gi 168812595 250 FPY RGLLL DTARNFFPTGEILRTIDA MAASKMNTF HWH VSDSQ SFP LRLDSA P.[12].VYTSD DVKTIVRHAKLR GIRV 335
gi 1346281 211 YPY RGILL DTARNFYSIDSIKRTIDA MAAVKLNTF HWH ITDSQ SFP LVLQKR P.[12].VYTKQ DIREVVEYGLER GVRV 296
gi 118367013 188 YPY RGLMI DTARHFLSVNTILKTIDS MQYNKLNVL HWH ITDDD SFP YPLQSF P.[12].QYSLT DIQYIVRYALLR GIQV 273
gi 24474977 187 YIY RGLMI DSARHFLSVETILKTIDS MLFNKLNVL HWH ITDTE SFP FPLKSF P.[12].QYSFE DIQYIVDQALNK GIQV 272
gi 62955499 172 FAF RGLLL DTSRHYLPLHAILKTLDA MAYSKFNVF HWH IVDDP SFP YQSRTF P.[13].IYTQS DVMRVIEHARMR GIRV 258
gi 31043932 178 YAF RGVMI DTARHYLPLNAILQTLDA MSYNKFNVL HWH IVDDQ SFP YVSDVY P.[13].IYTRE DIAAVIEFARLR GIRV 264
gi 21392072 235 FSH RGVLL DTARNFVPLKFIRSTLDA MAASKLNVL HWH VVDTH SFP LEITRV P.[12].TYSRQ DALNLVKYARLR GIRI 320
Feature 1
1NP0_B 237 LPEFDTPGHTLSWGKGQ .[17]. GPIN PTLNTTYSFLTTFFKEISEVF .[2]. QFI HLGGDEVEFK CW.[3]. PKIQDFM 322
query 253 LPEIDMPAHTGSWAEAY .[26]. GQLN PSIPKTYEVVKNVIQGTIAMF .[2]. SLF HGGADE INSD CW.[3]. LSVQKFV 347
gi 24653074 362 IPEIDAPAHAGNGWDWG .[26]. GQLN PKNNYTYLILQRIYEELLQHT .[3]. DFF HLGGDEVNLD CW.[1]. QYFNDTD 455
gi 168812595 336 LLEVDAPAHVGRAWGWG .[26]. GQLN PRNPHVYDLLQRIYAEILALT .[3]. DVF HLGGDEVSER CW.[1]. QHFNDTD 429
gi 1346281 297 LPEFDAPAHVGEGWQDT .[20]. GQLN PTKEELYDYLEDIYVEMAEAF .[3]. DMF HMGGDEVSER CW.[3]. EEIQNFM 386
gi 118367013 274 VPEIDSPGHAFSWGKSP .[13]. GQLD PSQKETWQLVNGVLTDLENQF .[3]. KYI HLGGDEVDEG CW.[3]. SDLKQYM 356
gi 24474977 273 IPEVDSPGHAFSWARSP .[13]. GQLD PTLNLTYTAVKGIMEDMNTQF .[3]. KYV HFGGDEVEEQ CW.[3]. PEIKEFM 355
gi 62955499 259 VPEFDSPGHTQSWGKGQ .[17]. GPVD PTVDTTYRFMERLLKEVKFVF .[2]. SYV HLGGDEVSFA CW.[3]. PSVGKFM 344
gi 31043932 265 IPEFDSPGHSTSWGKGQ .[17]. GPIN PTLNSTYTFVKNLFGDVKQVF .[2]. NYI HLGGDEVQFN CW.[3]. PNITKWM 350
gi 21392072 321 LIEIDGPSHAGNGWQWG .[26]. GQLN PLNDHMYAVLKEIFEDVAEVG .[3]. ETL HMGGDEVFLP CW.[3]. DEIRDGM 416
Feature 1
1NP0_B 323 .[ 9]. KKLESFYIQKVLDIIAT .[ 3]. GSIV WQE.[11]. TIVEV WKD .[6]. LSRVTASGFPVILS.[2]. WYLDLI 405
query 348 .[ 6]. SQLLEKFINNTLPEILS .[ 3]. TVVY WED.[18]. VIMQT WNN .[4]. TKQLVTSGYRVIVS.[4]. YYLDCG 434
gi 24653074 456 .[ 2]. GLWCDFMLQAMARLKLA .[ 7]. HVAV WSS.[12]. FTVQV WGG .[5]. NYDLLDNGYNVIFS.[4]. WYLDCG 537
gi 168812595 430 .[ 2]. DLWLEFTRRALHALERA .[ 7]. LVLL WSS.[15]. LGVQV WGS .[5]. SRAVLDAGFRSVLS.[4]. WYLDCG 514
gi 1346281 387 .[12]. LKLWNYFQKNAQDRAYK .[ 6]. PLIL WTS .[16]. YIIQV WTT .[5]. IQGLLQKGYRLIMS.[4]. LYFDCG 481
gi 118367013 357 .[ 8]. DDLQTFYRQTQKNLYRK .[ 5]. PAIY WSD.[11]. DIVQW WGE .[3]. FKLISNITNRIILS.[4]. AYLDVG 439
gi 24474977 356 .[ 8]. TDLQNYYRKNQVNIWKS .[ 5]. PAIF WAD.[ 9]. DIIQW WGS.[3]. FSSIKDLPNKIILS.[4]. TYLDVG 436
gi 62955499 345 .[ 9]. TKLESFYMESIMNITAA .[ 3]. TSIV WQD.[11]. TVLEI WKG .[6]. LSKMTKAGHRVLLS.[2]. WYINHI 427
gi 31043932 351 .[ 9]. SKLEQVYIQNVIDISET .[ 3]. SYIV WQE.[11]. TVVEV WKN .[6]. VAKVTAMGLRAIVS.[2]. WYLNII 433
gi 21392072 417 .[12]. LRLWSQFHQRNLNAWDE .[13]. SVII WSS.[16]. FIIQT WVE.[5]. NRELLQRGYRLIVS.[4]. WYLDHG 518
Feature 1
1NP0_B 406 .[ 5]. WRKYYKVEPL .[11]. FI GGEACL WGEYVDATNLTP RLWPRASAVG ERLWS.[12]. RLTRHRCRMVERG491
query 435 .[24]. WCGPFKTWET .[17]. VIGGEVAL WSEQADSTVMDS RIWPRAS AMAEAL WS.[16]. RLNEWRYRMVSRG549
gi 24653074 538 .[10]. ACAPYRTWQN .[19]. VLGGEVCM WTEQVDENQLDN RLWPRTA ALAERL WT.[17]. RISLFRNRLVELG641
gi 168812595 515 .[10]. HCGPYRSWQQ .[18]. VEGGAACQ WTEQLAAGGLDA RVWPRAA ALAERL WS.[12]. RLDTQRARLLARG612
gi 1346281 482 .[10]. WCSPYIGGQK .[16]. ILGGEVAL WSEQSDPATLDG RLWPRAA AFAERM WA.[11]. RMLHVRERLVRMG576
gi 118367013 440 .[14]. WKAMYAFNPQ .[ 7]. II GAEVCL WSELSDDDVYLT RIWTRTSAFS ERLWN.[16]. RMVFMKNRLNARG534
gi 24474977 437 .[ 8]. YGSMYNWDVL .[13]. IL GGETCL WSEMNDDSTQFQ RLWTRNSAFA ERLWN.[16]. RMVFMQHRLTARG531
gi 62955499 428 .[ 5]. WRNSYAVQPQ .[11]. VI GGEVAM WGEYVDATNLNP RLWPRACAAA ERLWS.[13]. RLEEFRCELVRRG514
gi 31043932 434 .[ 5]. WHKYYQYDPS .[11]. VM GGEACI WGEYVDATNLSP RLWPRASAVA ERLWS.[12]. RLDQQRCRMIRRG519
gi 21392072 519 .[ 9]. WRTVYSSGMP .[ 7]. VLGGEVCM WSEYVDQNSLES RIWPRAGAAAERM WS.[11]. RFYRYRERLLARG603
#
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F : Sequence alignment of 𝛽-Hex-Sl with nine other sequences by CD search. e amino acid residues Arg(), Asp(), His(),
Asp(), Glu(), Trp(), Trp(), Tyr(), Asp(), Trp(), and Glu() were predicted to be responsible for the activity of 𝛽-
Hex-S. e conserved amino acids are shown as yellow color.
genome consists of duplicated segments []. More than
gene duplication events have been detected by phylogenetic
analysis of plant, animal, and fungi before the separation
of three major eukaryotic lineages []. Specically, copy
numbers for genes with highly conserved functions seem to
be more stable than the number of genes with more divergent
functions. beta-Hexosaminidases from each kingdom (plant,
animal, and insect) are separated into two clades (clusters)
and each clade contains at least one member. Human genome
data analysis showed that both genes, HexA and HexB, are
located in dierent locus in the chromosomes-q- and
-q, respectively. ey are originated by gene duplication
[]. Most of the higher eukaryotes contain two or more
genes for the hexosaminidases. For example, Arabidopsis
thaliana contains Hex, Hex, and Hex []. Likewise
Drosophila melanogaster has three genes, Hexo, Hexo, and
fdl for hexosaminidase isoenzymes []. Even these proteins
are also located in dierent organelles. It has been reported
that some legume species have at least two Adh gene loci
and resulted from relatively ancient duplication events [].
From the accumulated evidences and phylogenetic topology,
it can be speculated that eukaryotic hexosaminidases might
be originated by multiple gene duplication, although more
experimental evidences are required to establish our hypoth-
esis.
Most of the prokaryotic and eukaryotic 𝛽-hexosamini-
dases reported so far play an important physiological role
in chitin recycling, a structural components of cell walls
[,,]. Plant 𝛽-hexosaminidases have been investigated in
a variety of tissues including seeds and leaves suggesting a role
in the storage of glycoproteins [–]. ey have also been
proposed to be involved in plants defense mechanisms and
reported as chitin-degrading enzymes [,]. A molecular
study of Arabidopsis 𝛽-hexosaminidases has shown that
e Scientic World Journal
T : Proteins sequences used for construction of phylogenetic studies.
SL GI number Name used in the tree Description Organism Taxonomy
. Homo sapiens-A beta-Hexosaminidase subunit-A,
HexA Homo sapiens Eukaryota
(Primates)
. Gorilla gorilla gorilla beta-Hexosaminidase subunit alpha
isoform Gorilla gorilla gorilla //
. Pongo abelii-A Predicted beta-hexosaminidase
Subunit-A Pongo abelii //
. Pan troglodytes-A beta-Hexosaminidase Subunit-A
isoform Pan troglodytes //
. Macaca mulatta-A beta-Hexosaminidase Subunit-A
precursor Macaca mulatta //
. Papio anubis-A beta-Hexosaminidase Subunit-A
isoform Papio anubis //
. Chlorocebus sabaeus-A beta-Hexosaminidase subunit alpha
isoform X Chlorocebus sabaeus //
. Callithrix jacchus-A beta-Hexosaminidase Subunit-A
isoform Callithrix jacchus //
. Tarsius syrichta-A beta-Hexosaminidase subunit-A
isoform X Tar s i u s s y r i c hta //
. Nomascus leucogenys-A Predicted beta-hexosaminidase
subunit-A Nomascus leucogenys //
. Homo sapiens-B beta-Hexosaminidase subunit-B,
HexB Homo sapiens //
. Pan troglodytes-B beta-Hexosaminidase subunit beta
isoform Pan troglodytes //
. Pongo abelii-B Predicted beta-hexosaminidase
subunit beta Pongo abelii //
. Chlorocebus sabaeus-B Predicted beta-hexosaminidase
subunit beta Chlorocebus sabaeus //
. Macaca mulatta-B beta-Hexosaminidase subunit beta Macaca mulatta //
. Papio Anubis-B Predicted beta-hexosaminidase
subunit beta Papio anubis //
. Callithrix jacchus-B beta-Hexosaminidase subunit beta
isoform Callithrix jacchus //
. Saimiri boliviensis-B Predicted beta-hexosaminidase
subunit beta Saimiri boliviensis //
. Ceratotherium simum-B Predicted beta-hexosaminidase
subunit beta Ceratotherium simum //
. Drosophila melanogaster- beta-Hexosaminidase, Hex1 Drosophila melanogaster Eukaryota
(Insect)
. Musca domestica beta-𝑁-Acetylglucosaminidase-like
isoform X Musca domestica //
. Ceratitis capitata beta-𝑁-Acetylglucosaminidase-like
isoform X Ceratitis capitata //
. Ceratitis capitata- beta-𝑁-Acetylglucosaminidase-like
isoform X Ceratitis capitata //
. Aedes aegypti beta-Hexosaminidase Aedes aeg ypti //
. Culex quinquefasciatus beta-𝑁-Acetylglucosaminidase C. quinquefasciatus //
. Spodoptera frugiperda Lysosomal beta-hexosaminidase Spodoptera frugiperda //
. Agrotis ipsilon beta-𝑁-Acetyl hexosaminidase Agrotis ipsilon //
. Trichoplusia ni beta-𝑁-Acetyl hexosaminidase Trichoplusia ni //
. Choristoneura fumiferana beta-𝑁-Acetyl hexosaminidase Choristoneura
fumiferana //
. Ostrinia furnacalis beta-𝑁-Acetylglucosaminidase Ostrinia furnacalis //
. Manduca sexta beta-𝑁-Acetylglucosaminidase Manduca sexta //
e Scientic World Journal
T : C ontinu e d .
SL GI number Name used in the tree Description Organism Taxonomy
. Drosophila melanogaster- beta-Hexosaminidase, Hex2 Drosophila melanogaster //
. Musca domestica beta-𝑁-Acetylglucosaminidase-like Musca domestica //
. Ceratitis capitata beta-𝑁-Acetylglucosaminidase-like Ceratitis capitata //
. Aedes aegypti beta-𝑁-Acetyl hexosaminidase Aedes aegypti //
. Tribolium castaneum beta-𝑁-Acetyl hexosaminidase
isoform X Tribol ium c ast ane um //
. Culex quinquefasciatus beta-𝑁-Acetylglucosaminidase-like C. quinquefasciatus //
. Ostrinia furnacalis beta-𝑁-Acetyl hexosaminidase Ostrinia furnacalis //
. Bombyx mori beta-𝑁-Acetyl hexosaminidase
precursor Bombyx mori //
. Solanum lycopersicum beta-Hexosaminidase1 Solanum lycopersicum Eukaryota
(planta)
. Solanum tuberosum Predicted beta-hexosaminidase -like Solanum tuberosum //
. Capsicum annuum beta-𝑁-Acetylhexosaminidase Capsicum annuum //
. Vitis vinifera Predicted beta-hexosaminidase-like Vitis v inifera //
. Cucumis sativus Predicted beta-hexosaminidase -like Cucumis sativus //
. Ricinus communis Putative beta-hexosaminidase Ricinus communis //
. Prunus persica beta-Hexosaminidase 2 Prunus persica //
Citrus sinensis Predicted beta-hexosaminidase -like Citrus sinensis //
. Arabidopsis thaliana beta-Hexosaminidase 2 Arabidopsis thaliana //
. Citrus sinensis Predicted beta-hexosaminidase -like Citrus sinensis //
. Glycine max Predicted beta-hexosaminidase -like Glycine max //
. Brachypodium distachyon Predicted beta-hexosaminidase -like Brachypodium
distachyon //
. Arabidopsis thaliana beta-Hexosaminidase 1 Arabidopsis thaliana //
. Eutrema salsugineum Hypothetical Protein Eutrema salsugineum //
. Cucumis sativus Predicted beta-hexosaminidase -like Cucumis sativus //
. Glycine max Predicted beta-hexosaminidase -like Glycine max //
. Prunus persica beta-Hexosaminidase Prunus persica //
. Solanum tuberosum Predicted beta-hexosaminidase -like Solanum tuberosum //
. Solanum lycopersicum Predicted beta-hexosaminidase Solanum lycopersicum //
. Brachypodium distachyon beta-Hexosaminidase subunit- B-like
isoform
Brachypodium
distachyon //
. Oryza brachyantha Predicted beta-hexosaminidase -like Oryza brachyantha //
. Oryza sativa Putative beta-hexosaminidase Oryza sativa //
. Aspergillus oryzae beta-𝑁-Acetylglucosaminidase Aspergillus oryzae Eukaryota
(Fungi)
. Aspergillus avus Putative
beta-𝑁-Acetylhexosaminidase Aspergillus avus //
. Aspergillus terreus Putative beta-hexosaminidase
precursor Aspergillus terreus //
. Neosartorya scheri Putative beta-hexosaminidase Neosartor ya scheri //
. Aspergillus niger Predicted 𝑁-acetylglucosaminidase Aspergillus niger //
. Aspergillus fumigatus Predicted
beta-𝑁-acetylhexosaminidase Aspergillus fumigatus //
. Aspergillus kawachii- beta-𝑁-Acetylhexosaminidase Aspergillus kawachii //
. Aspergillus clavatus Putative beta-𝑁-acetylhexosaminidase Aspergillus clavatus //
. Aspergillus kawachii- beta-𝑁-Acetylhexosaminidase
precursor Aspergillus kawachii //
. Penicillium oxalicum Putative
beta-,-𝑁-acetylglucosaminidase Penicillium oxalicum //
. Byssochlamys spectabilis Putative beta-𝑁-acetylhexosaminidase Byssochlamys spectabilis //
e Scientic World Journal
T : C ontinu e d .
SL GI number Name used in the tree Description Organism Taxonomy
. Streptomyces Plicatus beta-𝑁-Acetylhexosaminidase,
SpHex Streptomyces Plicatus Prokaryote
(Bacteria)
. Streptomyces coelicoavus Predicted beta-hexosaminidase Streptomyces
coelicoavus //
. Streptomyces lividans Putative beta-hexosaminidase
precursor Streptomyces lividans //
. Streptomyces
viridochromogenes Putative beta-hexosaminidase Streptomyces
viridochromogenes //
. Streptomyces coelicolor Putative beta-hexosaminidase Streptomyces coelicolor //
. Streptomyces olindensis Predicted beta-hexosaminidase Streptomyces olindensis //
. Streptomyces gancidicus Predicted beta-hexosaminidase S. gancidicus //
. Streptomyces coelicolor beta-Hexosaminidase Streptomyces Coelicolor //
. Streptomyces davawensis beta-𝑁-Acetylhexosaminidase Streptomyces davawensis //
. Streptomyces
viridochromogenes beta-𝑁-Acetylhexosaminidase Streptomyces
viridochromogenes //
. ermolum pendens Glycoside hydrolase family protein ermolum pendens Archea
Bold font indicates the experimentally characterized beta-𝑁-acetylhexosaminidases.
HEXO participates in 𝑁-glycan trimming in the vacuole,
whereas HEXO and/or HEXO could be responsible for the
processing of 𝑁-glycans present on secretory glycoproteins
[]. e 𝛽-Hex is also present at high levels during the
ripening of many fruits, including the climacteric fruit
tomato []andmango[]. Recently, it has been reported
that suppression of 𝑁-glycan processing enzymes increases
the shelf life of tomato fruits and capsicum [,]. e
𝛽-Hex, a cell wall enzyme, cleaves the terminal 𝑁-acetyl-
D-hexosamine residues and generates the paucimannosidic
𝑁-glycans present in most plant glycoproteins which in turn
downregulate the genes that encode for certain cell wall
degrading proteins, such as pectin methylesterase, glucan
endo-,-𝛽-D-glucosidase, 𝛽-,-glucanase, endoxyloglucan
transferase, pectinesterase, expansin, pectinacetylesterase, 𝛼-
galactosidase, pectate lyase, (-)-𝛽-mannan endohydrolase,
and 𝛽-galactosidase []. erefore, suppression of 𝛽-Hex
activity in transgenic fruits not only inhibited 𝑁-glycoprotein
degradation but also aects cellulose, hemicellulose, and
pectin degradation. Altogether, our phylogenetic analysis of
various GH 𝛽-Hexosaminidases with their comparative
functional properties suggests that plant 𝛽-Hexosaminidases
are cell wall bound enzymes derived from common bacterial
ancestor through multiple gene duplications and are involved
in 𝑁-glycan degradation or processing.
3.3. Resolved Predicted 3D Structure and Function. e
SWISS-MODEL web server [] was used to identify
thenowastemplatestructureforhomologymodeling
with .% the target-template sequence identity. Another
online server ModWeb Comparative Modeling Server ver-
sion SVN.r: M and I-TASSER []werealsoused
for further modeling for appropriate model selection. To
obtain an accurate homology model, it is very important
that appropriate steps are built into the process to assess
the quality of the model. erefore, the accuracies of the pre-
dicted models were checked through a series of tests such as
DFire [], QMEAN [], PROCHECK [], WHAT CHECK
[], VERIFY D [],andalsoModEvalModelevaluation
server []. A high quality predicted model was obtained
from ModWeb comparative modeling web server through
the analysis of predicted structures when compared with
ea ch ot her. Howeve r, the data for th e rest of model ed
structures are not shown. e Dre energy and QMEAN
score of best model were −. and ., respectively. e
Ramachandran plot showed .% of the residues in the
most favoured region, .% in the additional allowed region,
.% in the generally allowed region, and only .% in the
unfavourable region (Figure ). Ramachandran 𝑍-score is
−. indicating how well the backbone conformations of all
residuesarecorrespondingtotheknownallowedareasinthe
Ramachandran plot and within expected ranges for a well-
rened structure. None of the individual amino acid residues
wasinabadpackagingregion.estructuralaverageforthe
second-generation quality control value is within the normal
range. All contacts average is −. and 𝑍-score is −.,
which were within the normal ranges. e Anolea, QMean
graph and DSSP (dene secondary structure of protein) of
modeled 𝛽-Hex-Sl obtained from the structural assessment
by Swiss-model workplace are shown in Figure .
e X-ray crystal structure of human 𝛽-hexosaminidase
started at position of its gene-translated protein sequence
[].However,theDmodeledstructureof𝛽-Hex-Sl started
at position of its amino acid sequence as N-terminal. An
overall structural model of 𝛽-Hex-Sl is shown in Figure (a),
which contains residues in structural parts, glycosyl
hydrolase b domain-I, and glycosyl hydrolase super-
family domain-II including the (𝛽/𝛼)8barrel in the middle
part. e (𝛽/𝛼)8barrel structure houses the active site within
loops extending from the C termini of the strands that con-
stitute the 𝛽-barrel. e homologous domains are found in
e Scientic World Journal
gi145241784_Aspergillus_niger
gi358375826_Aspergillus_kawachii1
gi358372216_Aspergillus_kawachii2
gi115491163_Aspergillus_terreus
gi119484544_Neosartorya_scheri
gi70983560_Aspergillus_fumigatus
gi525585306_Penicillium_oxalicum
gi557727225_Byssochlamys_spectabilis
gi121719823_Aspergillus_clavatus
gi169766420_Aspergillus_oryzae
gi238483137_Aspergillus_avus
gi157106934_Aedes_aegypti1
gi170057261_Culex_quinquefasciatus1
gi294988604_Agrotis_ipsilon
gi508082176_Spodoptera_frugiperda
gi62722476_Choristoneura_fumiferana
gi114842947_Ostrinia_furnacalis1
gi37678109_Manduca_sexta
gi19072855_Trichoplusia_ni
gi498931058_Ceratitis_capitataX1-1
gi17647501_Drosophila_melanogaster-1
gi498964043_Ceratitis_capitata1
gi557771663_Musca_domestica1
gi642910295_Tribolium_castaneum
gi499003284_Ceratitis_capitata2
gi17933586_Drosophila_melanogaster-2
gi557764625_Musca_domestica2
gi157117066_Aedes_aegypti2
gi170029661_Culex_quinquefasciatus2
gi157804574_Ostrinia_furnacalis2
gi145651816_Bombyx_mori
gi357116549_Brachypodium_distachyon2
gi15220590_Arabidopsis_thaliana2
gi568879684_Citrus_sinensis3
gi255581813_Ricinus_communis
gi225450263_Vitis_vinifera2
gi440355382_Prunus_persica2
gi449532074_Cucumis_sativus2
gi568858509_Citrus_sinensis2
gi315440799_Capsicum_annuum2
gi350540008_Solanum_lycopersicum2
gi565386664_Solanum_tuberosum2
gi356528621_Glycine_max2
gi_357134815_Brachypodium_distachyon1
gi573945166_Oryza_brachyantha
gi115461737_Oryza_sativa
gi356568953_Glycine_max1
gi565358237_Solanum_tuberosum1
gi350538741_Solanum_lycopersicum1
gi401065909_Prunus_persica1
gi449459940_Cucumis_sativus1
gi30694211_Arabidopsis_thaliana1
gi567186303_Eutrema_salsugineum
gi478492476_Ceratotherium_simum-B
gi635028815_Chlorocebus_sabaeus-B
gi388454685_Macaca_mulatta-B
gi402871850_Papio_Anubis-B
gi297675458_Pongo_abelii-B
gi867691_Homo_sapiens-B
gi296194339_Callithrix_jacchus-B
gi403256462_Saimiri_boliviensis-B
gi114599673_Pan_troglodytes-B
gi332844225_Pan_troglodytes-A
gi640780361_Tarsius_syrichta-A
gi296213630_Callithrix_jacchus-A
gi441617200_Nomascus_leucogenys-A
gi402874775_Papio_anubis-A
gi635134633_Chlorocebus_sabaeus-A
gi387849165_Macaca_mulatta-A
gi1329112561_Pongo_abelii-A
gi4261632_Homo_sapiens-A
gi426379627_Gorilla_gorilla_gorilla
gi13786695_Streptomyces_Plicatus
gi494714113_Streptomyces_coelicoavus
gi511095822_Streptomyces_lividans
gi499338878_Streptomyces_coelicolor1
gi594145706_Streptomyces_coelicolor2
gi493092893_Streptomyces_gancidicus
gi640930344_Streptomyces_olindensis
gi490088482_Streptomyces_viridochromogenes2
gi490099150_Streptomyces_viridochromogenes1
gi505473521_Streptomyces_davawensis
gi119720203_ermolum_pendens
0.4
0.85
0.77
0.25
0.15
0.28
0.26
1
0.2
0.11
1
0.81
0.82
1
0.54
1
0.44
0.23
0.42
0.66
0.77
0.8
0.79
0.21
0.59
0.41
0.86
10.83
0.96
0.98
0.67
0.99
0.76
0.24
0.2
0.2
0.44
0.06
0.01
0.06
1
0.71
0.83
0.99
0.6
0.74
0.72
0.33
0.36
0.97
0.41
1
0.65
0.91
0.99
0.54
0.17
0.11
0.58
0.68
0.68
1
0.08
0.02
0
0
0
0.01
0.02
0.04
0.99
0.55
0.22
0.53
0.99
0.98
0.96
0.69
0.51
1.
Fungi clade (chitinase activities)
Insect clade-I (Hexo1)
Insect clade-II (Hexo2)
Plant clade-II (Hex2)
Dicot
Monocot
Plant clade-I (Hex1)
HexB
HexA
(Lysosomal N-glycan degrading)
Animal clade
Bacteria clade
(Peptidoglycan
degrading)
N-glycan processing
Chitinase activities
(Archea)
F : e phylogenetic tree based on beta-hexosaminidase amino acid sequences obtained by the maximum likelihood method.
ermolum (Archea) was used as an outgroup to reconstruct the phylogenetic tree. e percentage of replicate trees in which the associated
taxa clustered together in the bootstrap test ( replicates) is shown next to the branches. All analyses were performed with the WAG amino
acid substitution model and invariable and gamma distributed site rate categories. Detailed information about the sequences is shown in
Tabl e .
e Scientic World Journal
PROCHECK
A
L
b
B
a
l
pb
b
0 45 90 135 180
0
45
90
135
180
Psi (deg)
ILE 80
ALA 84
ILE 161
PHE 279
GLN 397
ASP 427
CYS 433
Phi (deg)
Plot statistics
Residues in most favoured regions [A, B, L] 400
Residues in additional allowed regions [a, b, l, p] 47
3
Residues in disallowed regions 4
Number of nonglycine and nonproline residues 454
Number of end-residues (excl. Gly and Pro) 2
Number of glycine residues (shown as triangles) 39
Number of proline residues 36
Total number of residues 531
−180 −135
−90
−45
−135
−90 −45
∼a
∼b∼p
∼l
∼b
∼b
∼b
88.1%
10.4%
0.7%
0.9%
—
—
—
100.0%
Inputatomonly
Ramachandran plot
Residues in generously allowed regions [∼a, ∼b, ∼l,∼p]
F : Ramachandran plot of the modeled structure of tomato
𝛽-𝑁-acetyl hexosaminidase provided by PROCHECK.
the crystal structure of S. plicatus (SpHEX) and S. marcescens
(SmCHB) [,]. An important secondary-structural motif
comprised helices and strands. e 𝛼-and𝛽-contents
of the modeled protein were found to be .% and
.%, respectively, as predicted by the program PROMOTIF
(Figure (a)). Structural similarity was further compared by
superimposition of modeled structure with template. e
modeled structure 𝛽-Hex-Sl closely resembled the template
structure (nowB) and it had good similarity with the
template upon superimposition (Figure (b)). e online D
ligand site prediction soware [] was used to identify the
ligand-binding site of the modeled structure 𝛽-Hex-Sl. e
amino acid residues Arg(), Asp(), His(), Asp(),
Glu(), Trp(), Trp(), Tyr(), Asp(), Trp(),
and Glu() were predicted to be present in the ligand-
biding site of 𝛽-Hex-Sl modeled structure (Figure (c)). e
space lled view of ligand-biding site of 𝛽-Hex-Sl with dock-
ing substrate 𝑁-acetyl-𝛽-D-glucosamine (NAG) is shown
in Figure (d). e COFACTOR online soware was used
to identify the functional motifs including ligand-binding
site, gene-ontology terms, and enzyme classication. e top
structural analogs of 𝛽-Hex-Sl modeled structure were
T : Top identied structural analogs in PDB by COFAC-
TOR.
Rank PDB Hit TM-score RMSDaIDENaCov.
nowB . . . .
gjxH . . . .
stA . . . .
csA . . . .
rcnA . . . .
hA . . . .
ghA . . . .
hpA . . . .
eplX . . . .
qba . . . .
TM-score is a measure of global structural similarity between query and
template protein.
RMSDais the RMSD between residues that are structurally aligned by TM-
align.
IDENaisthepercentagesequenceidentityin the structurally aligned region.
Cov. represents the coverage of the alignment by TM-align and is equal to
the number of structurally aligned residues divided by length of the query
protein.
identied in the protein data bank (Tab l e ). e nowB,
which had the TM-score . and RMSD ., was found
to be the top ranked among the various the homologous
proteins analyzed (Table ). e results indicated that our
predicted model structure of 𝛽-Hex-Sl was good, accurate,
and reliable.
e COFACTOR identied 𝛽-Hex-Sl with the classi-
cation EC... and predicted that amino acid residues
Asp() and Glu() could play important role in enzy-
matic reaction (Tabl e ).Itwasalsousedtosearchother
known homologous binding to compare the consensus bind-
ing with predicted ligand binding site. e three proteins
(lmyA, gkG, gjx) were found to have similar consensus
binding sites that were identical to the previously predicted
ligand-binding sites (Tab l e ). To predict the functions of
modeled structure of 𝛽-Hex-Sl, we used COFACTOR and
identied gene ontology (GO) terms. e consensus
prediction of GO terms and their GO-scores are shown in
Table .Tabl e shows a consistence of function (GO terms)
amongst top scoring templates. e GO score associated
with each prediction is dened as the average weight of
the GO term, where the weights are assigned based on
CscoreGO of the template from which the GO term is derived.
e most striking features for 𝛽-Hex-Sl described by GO
terms are homodimerization activities and localization in
cell membrane. In humans, two major 𝛽-hexosaminidase
isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer
of subunits 𝛼and 𝛽(% identity), whereas Hex B is a
homodimer of 𝛽subunits []. e molecular weight of
puried 𝛽-Hex-Sl as determined by gel-ltration (native
condition) also showed about four times greater value than
that determined by SDS-PAGE (denaturation condition) [].
ishappenedduetothedissociationoffoursubunitsfrom
each other by denaturing agent like SDS. e 𝛽-Hex-Sl
modeled D structure is a single chain protein containing
e Scientic World Journal
SSEEEEE SSSTHHHHHTHHHHHHHHHHHTHHHHHH HHHHHHHHHS EEEEEHHHHHTTT SSS
HHHHHSSS
EE
SEE TT TT TTSHHHHHHHHHHHHTT
20
10
10
8
6
4
2
0
0
1 112131415161718191101111
121 131 141 151 161 171 181 191 201 211 221 231
241 251 261 271 281 291 301 311 321 331 341 351
361
481 491 501 511 521 530
371 381 391 401 411 421 431 441 451 461 471
−20
−10
−40
−30
SSSEEEEESSSSSTTS EEEEEESSHHHHHHHHHHHHHHSSS EEEE SS SSS
Anolea
Qmean
dssp
20
10
10
8
6
4
2
0
0
−20
−10
−40
−30
Anolea
Qmean
dssp
20
10
10
8
6
4
2
0
0
−20
−10
−40
−30
Anolea
Qmean
dssp
20
10
10
8
6
4
2
0
0
−20
−10
−40
−30
Anolea
Qmean
dssp
20
10
10
8
6
4
2
0
0
−20
−10
−40
−30
Anolea
Qmean
dssp
LWPNPKSIFLSTNFTISHPYHRYLTPAVDRYRHLILSEHHRPIITPAINLTSSIPLQSLVISVSDVTSPLAHGVNESYSLSTPSDGSASAYISAATVWGAMRGLETFSQLVYGNPTRVSA
GVYIHDLPIFTHRGVMLDTSRNFYGVDHLLRLIKAMSMNKLNVFHWHITDSHSFPLVIPSEPELAGKGAYSNEMMYSPADVQKIVEYGMEHGVRVLPEIDMPAHTGSWAEAYPEIVTCAN
MFWWPAGSSPALAAEPGTGQLNPSIPKTYEVVKNVIQGTIAMFPDSLFHGGADEINSDCWNTDLSVQKFVASNGTLSQLLEKFINNTLPEILSLNRTVVYWEDVILSGNVKVNPSLLPPQ
S B BTTBHHHHHHHSS SSS B HHHHHHHHHHHHHTT EEEEEEEESSS HHHHHHSTT BSS
SEEEEEEESSSS HHHHHHHHHHHHHHT EEEEE BTTB
EEEE
SSS S S SSSSSS SB TT HHHHHHHHHHHHHHHHH
NVIMQTWNNGPNNTKQLVTSGYRVIVSSADYYYLDCGHGSFVGNDSRYDQPPGTDQGNGGSWCGPFKTWETIYNYDITYGLTDEEAPLVIGGEVALWSEQADSTVMDSRIWPRASAMAEA
HH SSSSTTSSTT HHHHHHHHHHHHHHHHHH STT TTS S
SS THHHHHHHHHT EEEE TTSSSBTTS SSS SSS SSSSSS HHHHHHH SS TTHHHHEEEEEEEE SSS TTTHHHHHTTHHHHHHHH
LWSGNRDETGMKRYAEATDRLNEWRYRMVSRGIGAESIQPLWCLKNPGMC
F : Anolea, Qmean, and DSSP (dene secondary structure of protein) obtained from the structural assessment by SWISS-MODEL
workplace online soware.
e Scientic World Journal
T : Top enzyme homologs in PDB by COFACTOR.
Rank CscoreEC PDB Hit TM-score RMSD IDEN Cov EC number Predicted active
site residues
. gjxA . . . . ... ,
. hpA . . . . ... ,
. ghA . . . . ... ,
. oaA . . . . ... ,
. yhtA . . . . ... ,
CscoreEC is the condence score for the enzyme classication (EC) number prediction. CscoreEC values range in between [-], where a higher score indicates
amorereliableECnumberprediction.
TM-score is a measure of global structural similarity between query and template protein.
RMSDais the RMSD between residues that are structurally aligned by TM-align.
IDENaisthepercentagesequenceidentityin the structurally aligned region.
Cov. represents the coverage of global structural alignment and is equal to the number of structurally aligned residues divided by length of the query protein.
𝛼-Helix
𝛽-Sheet
Glycosyl
hydrolase
20b domain
Glycosyl hydrolase
20 superfamily
domain
(a) (b)
(c)
NAG
(d)
F : e molecular D modeling of tomato beta-𝑁-acetyl hexosaminidase (𝛽-Hex-Sl). SPDB viewer and Chimera were used to prepare
the images. (a) e predicted D modeled structure is shown as ribbon diagram. e structure contains two fold domains (I and II) including
𝛼-helix (red), 𝛽-pleated sheets (purple), and coils (gray) e catalytic domain II is a (𝛽/𝛼)8barrel with the active site located at the C terminus
of the barrel. Template used for building this structure was now B(PDB). (b) Superimposition magic t image of the modeled structure
𝛽-Hex-Sl (blue) with template structure human now, human 𝛽-𝑁-acetyl-hexosaminidase (red), and human 𝛽-hexosaminidase B-subunit.
(c) e predicted ligand-binding site (active site) residues identied are depicted by as blue color. (d) Space lled view of ligand biding site
of 𝛽-Hex-Sl with docking substrate 𝑁-acetyl-𝛽-D-glucosamine (NAG).
e Scientic World Journal
T : Template proteins with similar binding sites searched by COFACTOR.
Rank CscoreLB PDB Hit TM-score RMSDaIDENaCov. BS-score Lig. Name Predicted binding sites
. lmyA . . . . . CP , , , , , ,
, , , ,
. gkG . . . . . NGT , , , , , ,
, ,
. gjx . . . . . Peptide
, , , , , ,
, , , , , ,
, ,
CscoreLB is the condence score of predicted binding site. CscoreLB values range in between [-], where a higher score is better site prediction.
BS-score is a measure of local similarity (sequence and structure) between template binding site and predicted binding site in the query structure. Based on
largescalebenchmarkinganalysis;wehaveobservedthataBS-score> reects a signicant local match between the predicted and template binding site.
TM-score is a measure of global structural similarity between query and template protein.
RMSDais the RMSD between residues that are structurally aligned by TM-align.
IDENaisthepercentagesequenceidentityin the structurally aligned region.
Cov. represents the coverage of global structural alignment and is equal to the number of structurally aligned residues.
T : Consensus prediction of gene ontology terms searched by COFACTOR.
Molecular function Biological process Cellular function
GO term GO score GO term GO score GO term GO score
GO: . GO: . GO: .
GO: . GO: . GO: .
GO: . GO: . GO: .
GO: . GO: . GO: .
GO: . GO: .
GO: .
GO: .
GO: .
GO: .
GO: .
Tab l e shows a consistence of function (GO terms) amongst top scoring templates. e GO score associated with each prediction is dened as the average
weight of the GO term, where the weights are assigned based on CscoreGO of the template from which the GO term is derived.
aminoacidsbutitdoesnothaveanyother-Hex-subunitlike
animals. Taken altogether our studies suggested that 𝛽-Hex-
Sl may need to exist as a homotetrameric structure during its
functional state and be located at the plant cell wall. Although
an involvement of 𝛽-Hex-Slinplantcellwallorfruitripening
has been reported recently [], depending on the properties
and behaviour of hexosaminidase homologues we could not
exclude the possibilities of their involvements in the other
physiological processes such as pathogenic resistance and
abiotic stress tolerance in plants.
4. Conclusion
We used the previously characterized 𝛽-hexosaminidases
and the novel putative 𝛽-hexosaminidase amino acid
sequences to reconstruct the phylogenetic tree. Phylogenetic
analysis placed 𝛽-Hex-Sl into the plant group, which might
originate from the common bacterial ancestral origin by
multiple gene duplications. Predicted D structure of 𝛽-
Hex-Sl contains amino acids with glycosyl hydrolase b
domain-I and glycosyl hydrolase superfamily domain-II
including the barrel (𝛽/𝛼)8in the central part. An impor-
tant secondary-structural motif comprised helices and
strands. e 𝛼-and𝛽-contents of the modeled protein were
found to be .% and .%, respectively. Eleven amino acids
werefoundtobeinvolvedinligand-bindingsiteof𝛽-Hex-
Sl. e amino acid residues Asp() and Glu() could
playimportantroleinenzyme-catalyzedreaction.efully
functional state of 𝛽-Hex-Sl needs to exist as a tetrameric
structureandbelocatedattheplantcellwall.epredicted
model provides a structural framework that can act as a guide
to develop a functional hypothesis to interpret experimental
data of 𝛽-𝑁-acetyl-D-hexosaminidases. ey may also facil-
itate eorts to design further site-directed mutagenesis to
explore the ligand recognition and the downstream signaling
mechanisms for the fruit ripening. e presented modeling
approach can be extended to other proteins as well.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
e Scientic World Journal
Acknowledgment
e authors are grateful to Abdul Wahab Abideen, a graduate
student, Department of Molecular Biology, FRST, UNIMAS,
for his help in correction of spelling, grammar, and punctua-
tion of this paper.
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