ORIGINAL PAPER

A hydrolytic c-glutamyl transpeptidase from thermo-acidophilicarchaeon Picrophilus torridus: binding pocket mutagenesisand transpeptidation

Rinky Rajput • Ved Vrat Verma • Vishal Chaudhary •

Rani Gupta

Received: 16 July 2012 / Accepted: 9 October 2012 / Published online: 27 October 2012

� Springer Japan 2012

Abstract c-Glutamyl transpeptidase of a thermo-acido-

philic archaeon Picrophilus torridus was cloned and

expressed using E. coli Rosetta-pET 51b(?) expression

system. The enzyme was expressed at 37 �C/200 rpm with

c-GT production of 1.99 U/mg protein after 3 h of IPTG

induction. It was improved nearby 10-fold corresponding

to 18.92 U/mg protein in the presence of 2 % hexadecane.

The enzyme was purified by Ni2?-NTA with a purification

fold of 3.6 and recovery of 61 %. It was synthesized as a

precursor heterodimeric protein of 47 kDa with two sub-

units of 30 kDa and 17 kDa, respectively, as revealed by

SDS-PAGE and western blot. The enzyme possesses

hydrolase activity with optima at pH 7.0 and 55 �C. It was

thermostable with a t1/2 of 1 h at 50 �C and 30 min at

60 �C, and retained 100 % activity at 45 �C even after

24 h. It was inhibited by azaserine and DON and PMSF.

Ptc-GT shared 37 % sequence identity and 53 % homology

with an extremophile c-GT from Thermoplasma acido-

philum. Functional residues identified by in silico approa-

ches were further validated by site-directed mutagenesis

where Tyr327 mutated by Asn327 introduced significant

transpeptidase activity.

Keywords Picrophilus torridus � Thermo-acidophile �c-Glutamyl transpeptidase � Expression � PET 51b(?) �Site-directed mutagenesis

Introduction

c-Glutamyl transpeptidases (c-GTs; EC 2.3.2.2) belong to

Ntn hydrolase superfamily which catalyzes the cleavage of

c-glutamyl linkage of glutathione (GSH) and subsequently

transfer their c-glutamyl moiety to peptides and amino acid

acceptors (Tate and Meister 1981). They are heterodimeric

proteins coded by a single gene which during the process of

autocleavage, is cleaved into larger and smaller subunit.

The N-terminal nucleophile of smaller subunit is threonine

and is responsible for both autoprocessing and catalysis

(Castellano and Merlino 2012). The enzyme is ubiquitous

in occurrence and is distributed in bacteria and mammals.

This enzyme is involved in glutathione metabolism by

cleaving the glutamyl amide bond and accounts for

intracellular cysteine pool (Pompella et al. 2007; Lee et al.

2004). Apart from its physiological importance, its

biotechnological importance has been recognized in the

synthesis of c-glutamyl peptides, pharmaceutical and food

applications and is also used for debittering of amino acids

(Suzuki et al. 2007). In this respects, several mesophilic

bacterial c-GTs from Bacillus sp. viz., B. licheniformis,

B. subtilis, B. pumilus, and a few thermophilic c-GTs both

from eubacteria and archaea have been biochemically

characterized (Lin et al. 2006; Shuai et al. 2011; Murty

et al. 2011; Castellano et al. 2010, 2011). Thermostable

enzymes are important as they have better activity and

stability. With the advent of whole genome database, it has

become feasible to tap extremophilic archaea for industrial

enzymes (Cavicchioli 2007). Till date, several enzymes

such as amylases, glucosidases and pantothenate kinases

have been characterized from Sulfolobus solfataricus and

Picrophilus torridus (Kim et al. 2004; Takagi et al. 2010).

We report functional expression, biochemical character-

ization and structural analysis of c-glutamyl transpeptidase

Communicated by H. Atomi.

R. Rajput � V. V. Verma � V. Chaudhary � R. Gupta (&)

Department of Microbiology, University of Delhi,

South Campus, New Delhi 110021, India

e-mail: ranigupta15@yahoo.com

123

Extremophiles (2013) 17:29–41

DOI 10.1007/s00792-012-0490-8

from thermo-acidophilic archaebacteria P. torridus. Based

on in silico study, identified functional residues were

confirmed by site-directed mutagenesis which introduced

transpeptidation.

Materials and methods

Cloning and expression vectors namely pGEM-T easy

vector and pET 51b(?) vector were purchased from

Promega Co., USA and Novagen, respectively. Taq DNA

polymerase, T4 DNA ligase and restriction enzymes

were purchased from New England Biolabs, Beverly,

MA. 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside

(X-Gal), isopropyl b-D-thio-galactopyranoside (IPTG) and

carbenicillin were purchased from Sigma-Aldrich Inc.,

USA. Ni2?-NTA purchased from G-Biosciences, USA.

Genomic DNA extraction, Plasmid extraction and gel

elution were performed using the Qiagen DNeasy Blood &

Tissue Kit, Mini prep and gel elution kits purchased from

Qiagen, Hilden, Germany. Recombinant E. coli was grown

at 37 �C in LB broth (10 g of tryptone, 5 g of yeast extract,

and 10 g of NaCl per liter, pH 7.0; Hi-media) supple-

mented with carbenicillin (50 lg/mL). E. coli DH5a and

E. coli Rosetta were used as cloning and expression host,

respectively.

Strain and growth conditions

Picrophilus torridus DSM 9790 was obtained from DSMZ,

Germany and was grown aerobically at 55 �C, 100 rpm at

pH 1.0 for 10 days according to specification from

DSMZ. This medium contained (per liter) KH2PO4 3 g;

MgSO4�7H20 0.5 g; CaCl2�2H2O 0.25 g; yeast extract 2 g;

glucose 10 g; (NH4)2 SO4 0.2 g and pH was adjusted with

concentrated H2SO4 to reach a pH of about 1.0.

Cloning and expression of ptc-gt

Genomic DNA was extracted from P. torridus using Qiagen

DNeasy Blood & Tissue Kit and used as template for PCR

amplification. Primers were designed from the ORF

PTO1185 of c-GT identified from the complete genome of

P. torridus PTO1185 available at NCBI. The sequence of

forward primer along with Kpn I site was 50-GGTACC

AATGTATATGAATTAT-30 and the reverse primer with

Sac I site was 50-GAGCTCATTAGATGATATTTTA-30.The sequence recognized by restriction enzymes was

underlined. The PCR condition consisted of an initial dena-

turation at 95 �C for 5 min followed by 30 cycles of 95 �C

for 30 s, 42.2 �C for 30 s and 72 �C for 2 min, and a final

extension of 10 min at 72 �C. The PCR product of 1.4 kb

was ligated with pGEM-T Easy and transformed into E. coli

DH5a competent cells. Positive colonies were screened by

colony PCR and reconfirmed by fallout analysis.

Expression vector, pET 51b was linearized by double

digestion with Kpn I/Sac I at 37 �C for 16 h followed by

gel elution. The 1.4 kb fallout was ligated overnight into

linearized pET 51b using T4 DNA ligase at 16 �C. The

resulting plasmid was transformed into E. coli Rosetta

cells. The recombinant E. coli with pET 51b-ptc-gt was

cultivated in LB medium supplemented with carbenicillin

(50 lg/mL) at 37 �C/200 rpm until the OD600nm reached

0.5 and then induced with 0.5 mM IPTG. After 3 h incu-

bation, samples were harvested and the cells were sepa-

rated by centrifugation at 74009g for 10 min. Harvested

1 g cell pellet was suspended in 10 mL ice cold 19 PBS

and lysed on ice by sonication at 2 s pulse on and 2 s pulse

off for 10 min, and the lysate clarified by centrifugation

and expression was checked in the clear lysate by c-GT

assay and SDS-PAGE analysis. E. coli Rosetta (DE3) cells

harboring only pET 51b without insert was processed in a

similar manner and taken as control. The expression was

also studied in the presence of hexadecane 1.0–4.0 %, v/v

in LB-carbenicillin medium under same conditions.

Purification of Ptc-GT

Purification of Ptc-GT was performed using Ni–NTA resin

for His-tagged fusion protein 69 HIS-Ptc-GT. The clear

lysate was loaded onto Ni2?-NTA resin (0.5 mL) and left

in the column for 30 min under mild shaking. The column

was washed with 10 column volumes of 50 mM phosphate

buffer, pH 7.5 with 300 mM NaCl and 10 mM imidazole.

Elution of bound protein was performed with 2 column

volume of 50 mM phosphate buffer, pH 7.5 with 300 mM

NaCl in a linear gradient of imidazole (50–500 mM). The

purified protein was then treated with 1 M guanidine

hydrochloride in 1:1 ratio for 30 min at room temperature

and total protein was estimated at 280 nm. The purity of

protein was checked by HPLC on C18 column (Shimadzu,

Japan) using acetonitrile:water (90:10) as mobile phase

with flow rate of 0.5 mL/min and protein was checked on

SDS-PAGE and western blot analysis.

Enzyme assay for c-GT activity

The enzymatic hydrolytic activity was measured using c-

glutamyl-p-nitroanilide (GpNA) as substrate according to

the procedure described by Tate and Meister (1985). The

assay mixture contained 1 mM GpNA and appropriately

diluted enzyme in 50 mM Tris–HCl pH 7.0. The reaction

was incubated at optimum temperature for 5 min and ter-

minated with 100 lL of 3 mM (v/v) acetic acid. The

release of p-nitroaniline was monitored at 410 nm. One

unit of c-GT activity is defined as the amount of enzyme

30 Extremophiles (2013) 17:29–41

123

that releases 1 lmol of p-nitroaniline per minute. Hydro-

lysis of GpNA in the absence of acceptor was considered as

hydrolytic activity and in the presence of acceptor (20 mM

glycyl-glycine) was taken as transpeptidase activity.

Protein estimation

The total protein was estimated spectrometrically by the

method of Bradford (1976) using bovine serum albumin

(BSA) as the standard.

Zymogram analysis of Ptc-GT (Ohlsson et al. 1986)

Purified protein was run on NATIVE-PAGE using 10 %

polyacrylamide gel. After electrophoresis, the gel was

washed with Tris–HCl buffer (50 mM, pH 7.0) for 10 min

on rocker. The gel slab was then overlaid onto agar plate

containing Tris–HCl buffer (50 mM, pH 7.0), mixed with

1 mM GpNA as substrate and incubated at 50 �C till yellow

band appeared. The gel having a yellow band was devel-

oped with 0.1 % (w/v) sodium nitrite in 1.0 M HCl for

5 min under mild shaking (50 rpm) at 25 ± 1 �C and

rinsed thoroughly with distilled water. For another 5 min,

0.5 % (w/v) ammonium sulphamate in 1.0 M HCl was

poured onto the gel and incubated under shaking at

25 ± 1 �C. Excess ammonium sulphamate was removed

by repeated washing with distilled water. At last, band was

treated with N-(1-naphthyl) ethylene diamine in 47.5 %

ethanol, till a pink band corresponding to the yellow band

appeared on GpNA agar plate.

Western blot analysis to detect Ptc-GT

Western blot analysis was performed using a modified

enzymatic chemiluminescence (ECL). The protein was

analyzed on 10 % SDS-PAGE and transferred to PVDF

membrane (Bio-Rad). The membrane was blocked in 3 %

skim milk for 1 h in PBS and proceeded separately for

strep tag and his tag.

Strep-tag detection for larger subunit

The primary antibody, strep protein (1:5000) was added and

the membrane was incubated for 1 h at mild shaking and

washed three times for 10 min with PBS with 0.1 % Tween

20 (PBST). The proteins were visualized with enhanced

chemiluminescence reagent, diaminobenzidine (0.1 % w/v)

solution prepared in hydrogen peroxide for 5 min.

His-tag detection for smaller subunit

Primary antibody, anti-his (1:5000) was added and the

membrane was incubated for 1 h at mild shaking and

washed three times for 10 min with PBS with 0.1 % Tween

20 (PBST). Secondary anti-rabbit antibody (1:5000) con-

jugated with alkaline phosphatase was then added, and

membrane was incubated for 1 h. The membrane was

washed three times with PBST. The proteins were visual-

ized with enhanced chemiluminescence reagents in alka-

line phosphate buffer.

Biochemical characterization of Ptc-GT

Effect of pH and temperature on activity and stability

of Ptc-GT

Effect of pH on c-GT activity was studied in the pH range

from 2.0 to 11.0 using overlapping buffers 50 mM of

Glycine–HCl (pH 2.0), citrate buffer (pH 3.0–6.0), citrate–

phosphate buffer (pH 3.0–7.0), phosphate buffer (pH

7.0–8.0), Tris–HCl buffer ((pH 7.0–9.0), glycine–NaOH

buffer and sodium carbonate-bicarbonate buffer (pH

9.0–11.0) and glycine-phosphate hydroxide buffer (pH

10.0–11.0). Similarly, the effect of temperature on c-GT

activity was determined at different temperatures ranging

from 30 to 80 �C at optimum pH. The activity was

expressed as the percentage relative activity with respect to

maximum activity which was considered as 100 %.

The stability of Ptc-GT was studied over a broad range

of pH and temperature by pre-incubating the enzyme in

buffers of 10 mM of varying pH (2.0–11.0) for 1 h at

25 ± 1 �C and at temperature from 30 to 80 �C for a

period of 2 h and also at 45 �C for 24 h. The enzyme

activity was expressed as the residual activity against the

control which was taken as 100 %.

Effect of inhibitors, reducing agents and metal ions

on the activity of Ptc-GT

Enzyme was pre-incubated with inhibitors viz., azaserine,

DON (6-diazo-4 oxo-norleucine), ethylene diamine

tetraacetic acid (EDTA), ethylene glycol tetraacetic acid

(EGTA), N-bromosuccinimide, iodoacetic acid, p-chlor-

omercuric benzoic acid, phenylmethylsulfonyl fluoride

(PMSF), 1,10-o-phenanthroline (Sigma-Aldrich, USA;

ICN chemicals, USA) and reducing agents namely

b-mercaptoethanol (b-ME) and dithiothreitol (DTT)

(Sigma-Aldrich USA; ICN chemicals, USA) at 25 ± 1 �C

at a final concentration of 5 mM and 10 mM for 15 min.

Similarly, the effect of metal ions was studied by pre-

incubating the enzyme with metal ions viz., Ba2?, Ca2?,

Cd2?, Co2?, Cu2?, Fe2?, Mg2?, Hg2?, Mn2?, Ni2?, Pb2?

and Zn2? at a final concentration of 5 mM and 10 mM for

1 h. Residual activity was measured under standard assay

conditions.

Extremophiles (2013) 17:29–41 31

123

In silico studies

Sequence comparison and phylogenetic analysis of Ptc-GT

Ptc-GT amino acid sequence was downloaded from

microbial genome database (Futterer et al. 2004). PSI-Blast

algorithm was used to create percentage sequence identity

and homology among all the c-GTs sequences including

Ptc-GT (Altschul et al. 1997). Multiple sequence alignment

was performed by ClustalW using default parameters, and

phylogenetic relationship was drawn using UPGMA algo-

rithm (Larkin et al. 2007).

Structural analysis of Ptc-GT

Ptc-GT amino acid sequence was submitted for PSI-Blast

against Protein Data Bank for suitable template selection,

and putative c-GT from Thermoplasma acidophilum

(Tac-GT, PDB code 2I3O) was selected as template for

homology modeling. Modeller 9.10 was used for homology

modeling for wild and mutant (Y327N) types (Eswar et al.

2006). Side chain modifications were done using SCWRL

tool and optimum bond length and bond angles were

attained by energy minimization using AMBER force field

(Canutescu et al. 2003; van der Spoel et al. 1994). Quality

of model was validated by Structural Analysis and Verifi-

cation Server (PROCHECK and WHATCHECK program)

(Laskowski et al. 1993; Hooft et al. 1996). Secondary

structural components were analyzed by PSIPRED and

VADAR server (McGuffin et al. 2000; Willard et al. 2003).

Pocket finder and CASTp were used for active site

predictions (Ruppert et al. 1997; Dundas et al. 2006).

Patchdock and Autodock programs were used for docking

study (Duhovny et al. 2005; Morris et al. 1998). Protparam

server was used for analyzing amino acid composition

(Gasteiger et al. 2005). PyMol, a protein viewer tool was

used for visualization and structural alignment of protein

(Seeliger and De Groot 2010).

Functional validation of active site residues present

in Ptc-GT by site-directed mutagenesis

Based on the structural analysis of larger and smaller

subunit of Ptc-GT, Tyr327, Arg346, Tyr349 and His368

were selected to perform site-directed mutation, where

these residues were replaced with Asn327, Glu327,

Ala346, Glu346, Asp349 and Ser368, respectively. Site-

directed mutagenesis was performed by QuickChangeTM

Site-Directed Mutagenesis Kit (Stratagene). PCR experi-

ment was set up with pET 51b-ptc-gt expression construct

using gene-specific primers according to the protocol pro-

vided with kit. PCR products were digested with the Dpn I,

to remove methylated wild-type plasmid, and 5 lL

amplicon was transformed into E. coli XL-10 cells. Colo-

nies obtained were grown in LB-amp and plasmids were

isolated. Mutated plasmids were then transformed in

expression host, E. coli Rosetta, and mutant proteins were

purified as described earlier for wild protein. The purified

proteins were then characterized kinetically where kinetic

parameters, Km, Vmax and Kcat/Km can be determined by

performing a state kinetics at varying concentrations of

c-glutamyl-p-nitroanilide (0.01–1.0 mM) under optimum

conditions. Kinetic parameters were calculated by

Lineweaver–Burk plot using Sigma Plot software. Com-

parative docking and fluorescence analysis (Varian Cary

Eclipse Fluorescence Spectrophotometer, USA) was done

for mutant Y327N and wild type to support site-directed

mutagenesis outcomes.

Statistical analysis of data

All the above experiments were repeated in triplicate, and

the final values have been presented as mean ± standard

deviation.

Results

Cloning and expression of ptc-gt from P. torridus

PCR amplification using the specific primers resulted in a

1.4 kb gene product which was ligated into pGEM-T Easy

and subcloned into pET 51b. It was transformed in E. coli

Rosetta cells. Expression was studied at 37 �C/200 rpm

with c-GT production of 1.99 U/mg protein after 3 h

of IPTG induction. Unprocessed precursor c-GT was

observed as a major protein of 47 kDa on SDS-PAGE

(Fig. 1a). The addition of 2 % n-hexadecane during pro-

duction enhanced c-GT production to nearly 10-fold with

18.92 U/mg protein after 3 h induction at 37 �C/200 rpm,

and SDS-PAGE showed that c-GT was completely pro-

cessed into mature protein constituted by two subunits,

large and small with apparent molecular mass of 30 kDa

and 17 kDa, respectively (Fig. 1a).

Purification of Ptc-GT

Ptc-GT was purified by Ni2?-NTA matrix with a purifi-

cation fold and recovery of 3.6 and 61 %, respectively. The

purity of purified protein was confirmed further by HPLC

on C18 column using UV detector (data not shown). The

protein was a heterodimer with subunits of 30 and 17 kDa

as revealed by SDS-PAGE and confirmed by western blot

analysis, the larger subunit as strep protein and the smaller

subunit as his protein (Fig. 2a). The activity of Ptc-GT was

confirmed by zymogram on GpNA agar plate where the

32 Extremophiles (2013) 17:29–41

123

released p-nitroaniline was diazotized with N-(1-naphthyl)

ethylenediamine to a red azo dye and developed a pink

zone of GpNA hydrolysis coinciding with the protein band

on the NATIVE-PAGE (Fig. 2b).

Biochemical properties of Ptc-GT

Effects of pH and temperature on the activity and stability

of Ptc-GT

Ptc-GT was active over a wide pH range, 4.0–9.0 with

[40 % of hydrolytic activity and optima at 7.0 (Fig. 3a).

The enzyme was active over temperature 40–60 �C with

[50 % activity having maxima at 55 �C (Fig. 2b).

The enzyme was stable within a pH range 3.0–10.0 with

[50 % residual activity (Fig. 3a). The protein was found

100 % stable at 45 �C after 24 h with a t1/2 of 1 h at 50 �C

and 30 min at 60 �C (Fig. 3b).

Effects of inhibitors, reducing agents and metal ions

on the activity and stability of Ptc-GT

Ptc-GT was completely inhibited by 1 mM azaserine,

DON (6-diazo-4 oxo-norleucine) and PMSF (data not

shown). It retained [90 % activity in the presence of

10 mM chelating agents like EDTA, EGTA and 1, 10-o-

phenanthroline and was not inhibited by N-bromosuccini-

mide and iodoacetic acid (data not shown). However,

reducing agents like DTT and b-mercaptoethanol have no

significant effect on the activity of the enzyme (data not

shown). The enzyme retained [90 % activity in the pres-

ence of most of divalent cations like Ba2?, Ca2?, Co2?,

Cd2?, Fe2?, Hg2?, Mg2?, Mn2? and Zn2?. However, it

retained 40 % activity in presence of Cu2? and Ni2? (data

not shown). Besides, mutant Y327N retained [90 %

transpeptidase activity in the presence of Ba2?, Ca2?,

Cu2?, Mg2?, Mn2?, and Zn2? and was completely inhib-

ited by Cd2?, Co2?, Fe2?, Hg2?, Ni2 and Pb2? (data not

shown).

Fig. 1 Expression profile of Ptc-GT after 3 h at 37 �C/200 rpm. a SDS-

PAGE profile: Lane 1 protein marker, Lane 2 pET 51b-Ptc-GT only,

Lane 3 pET 51b-Ptc-GT ? IPTG (0.5 mM), Lane 4 pET 51b-

Ptc-GT ? IPTG ? hexadecane (1 %), Lane 5 pET 51b-Ptc-GT ?

IPTG? hexadecane (2 %), Lane 6 pET 51b-Ptc-GT ? IPTG ? hexa-

decane (3 %), Lane 7 pET 51b-Ptc-GT ? IPTG ? hexadecane (4 %).

b Data were analyzed using Student’s t test where filled bars havingalphabets (a, b, c) are statistically different

Fig. 2 Purification profile of Ptc-GT. a SDS-PAGE profile and

western blot of purified protein: Lane 1 protein marker, Lane 2 pET

51b-Ptc-GT only, Lane 3 pET 51b-Ptc-GT ? IPTG (0.5 mM), Lane4 purified Ptc-GT on Ni2?-NTA, Lane 5 strep-tag blot of pET 51b-

Ptc-GT only, Lane 6 strep-tag blot of Ptc-GT, Lane 7 his-tag blot of

pET 51b-Ptc-GT only Lane 8 his-tag blot of Ptc-GT. b Zymogram of

Ptc-GT on NATIVE-PAGE: Lane 1 pET 51b-Ptc-GT only, Lane 2Ptc-GT, Lane 3 zymogram of pET 51b-Ptc-GT only, Lane 4zymogram of Ptc-GT

Extremophiles (2013) 17:29–41 33

123

Comparative sequence analysis of Ptc-GT with other

bacterial and eukaryotic c-GTs

Comparative sequence analysis of Ptc-GT with other bacte-

rial and eukaryotic c-GTs revealed that Ptc-GT does not

share significant identity and homology with any of the

c-GTs (Table 1). It shared the highest sequence identity

of 37 % and homology of 53 % with an extremophile

Thermoplasma acidophilum (data not shown). Further, phy-

logenetic analysis revealed three distinct clusters of c-GTs

belonging to non-extremophiles, eukaryotes and then to extr-

emophiles. Among these, Ptc-GT was found closely related

to c-GTs of other extremophiles and thermophiles (Fig. 4).

Multiple sequence alignment of Ptc-GT with other ex-

tremophiles, thermophiles and E. coli c-GTs revealed con-

served primary and secondary structure among all c-GTs

(Fig. 5). The putative internal cleavage site for large and

small subunit of Ptc-GT was predicted as Thr308, conserved

among all the c-GTs. The most critical difference of the

Ptc-GT when compared with E. coli c-GT (Ecc-GT) was

absence of lid loop in comparison to Ecc-GT (residues P438-

G449) (Okada et al. 2006). Further, c-glutamyl binding

moiety residues Thr409, Gly483, Gly484 of E. coli were

found conserved in Ptc-GT as Gly387, Gly388 (oxyanion

hole residues) whereas Thr409 is replaced by Ser325 in

Ptc-GT similar to other extremophiles (Ruepp et al. 2000;

Boanca et al. 2007). Other active residues known for Ecc-GT

were Arg114, Asn411, Gln430, Asp433, Leu461, Ser462,

Ser463, Pro482 and Ile487 were different in Ptc-GT as Ser68,

Tyr327, Arg346, Tyr349, Phe367, His368, Thr369, Met386

and Gln391, respectively. However, these binding residues of

Ptc-GT were observed to be conserved and semi-conserved

in c-GTs from other extremophiles and thermophiles.

Structural analysis of Ptc-GT

Model of Ptc-GT was built to identify structural basis that

could be accounted for comparing functional properties of

pH

1 2 3 4 5 6 7 8 9 10 11 12

Rel

ativ

e ac

tivity

(%

)

0

20

40

60

80

100

120

pH

Res

idua

l act

ivity

(%

)

Temperature (°C)

25 30 35 40 45 50 55 60 65 70 75

Rel

ativ

e ac

tivity

(%

)

0 20 40 60 80 100 120

Res

idua

l act

ivity

(%

)

45°C50°C60°C70°C

Ptγ-GT was 100% at 45°C after 24 h

0

20

40

60

80

100

120

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 120

20

40

60

80

100

120

Temperature (°C)

A

B

Fig. 3 Effect of pH (a) and temperature (b) on the activity of Ptc-GT. 100 % Activity corresponds to 33 U/mg protein

34 Extremophiles (2013) 17:29–41

123

Ptc-GT with other c-GTs. As Tac-GT showed the highest

sequence identity with Ptc-GT, it was selected as template

for homology modeling. Crystal structure of Tac-GT has

been available in Protein Data Bank but description has not

been reported in literature. The stereochemical quality of

final model showed that 89.6 % residues positioned in most

favorable region and 0.0 % residues positioned in disal-

lowed region of Ramachandran plot. Root mean square

deviation (RMSD) value in the Ca positions between

template and modeled structure was observed as 1.504 A.

Similar to crystal structures of c-GT from Ecc-GT and

Tac-GT, the modeled structure of Ptc-GT also showed a

abba-core structure where central b-sheets were sand-

wiched by a-helices. Modeled structure of Ptc-GT was

selected as primary source for further analysis (Fig. 6a).

Identified active site of Ptc-GT was cross validated by

comparing modeled structure of Ptc-GT with crystal structure

of Ecc-GT. c-Glutamyl moiety was docked into active site of

Ptc-GT model. Stereoview of the Ptc-GT enzyme complex

with glutathione showed that this substrate is perfectly docked

Table 1 Parameters related to the amino acid composition of

P. torridus compared with E. coli and G. thermodenitrificans

Amino acid parameters Ptc-GT Ecc-GT Gtc-GT

Number of protein residues 478 580 534

Molecular weight (Da) 53,279.1 61,767.9 57,668.5

Total number of uncharged polarresidues Gln ? Asn ? Thr ? Ser(%)

22.5 20.4 15.3

Total number of hydrophobicresidues (Ala ? Val ? Ile ? Leu) (%)

27.9 31 31.9

Number of Arg (R) (%) 2.9 2.8 4.1

Number of Lys (K) (%) 6.1 6.7 3.7

Arg/Lys 0.48 0.42 1.11

Arg/(Arg ? Lys) 0.32 0.29 0.53

Total number of positively chargedresidues (Arg ? Lys) (%)

9.0 9.5 7.8

Total number of negatively chargedresidues (Asp ? Glu) (%)

10.6 11.4 10.1

(Asp ? Glu)/(Arg ? Lys) 1.23 1.2 1.29

Total number of charged residues(Glu ? Asp ? Lys ? Arg) (%)

19.6 20.9 17.9

Total number of Gly (%) 8.6 9.7 10.7

Total number of Pro (%) 3.8 6.0 7.7

Fig. 4 Phylogram of c-GTs.

The amino acid sequences of

c-GTs were used from

Escherichia coli (NP417904),

Salmonella enterica(YP005244539), Francisellatularensis (YP005318273),

Helicobacter pylori(NP207909), Yersinia pestis(NP668315), Burkholderiamallei (YP105670), Bacillussubtilis (NP389723),

Staphylococcus aureus(YP185087), Bacillus anthracis(YP002860670), Pig

(NM214030), Human

(NM005265), Rat (NM053840),

Mouse (Q60928), Bacillushalodurans (NP241733),

Geobacillus thermodenitrificans(YP001127364), Deinococcusradiodurans (Q9RU70),

Thermus thermophilus(YP004378), Picrophilustorridus (YP023963),

Thermoplasma acidophilum(NP394454). Online available

ClustalW tool and UPGMA

algorithm with default

parameters was used for

phylogenetic prediction

Extremophiles (2013) 17:29–41 35

123

inside the cavity of active site. Binding cavity of Ptc-GT was

empty because of the lack of lid loop toward the active site of

Ptc-GT, but it was observed in Ecc-GT. All the residues

proposed to be a part of binding site, whereas the active

residues for acceptor binding site produced by multiple

sequence alignment were found in the cavity (Fig. 6b).

Functional validation of active side residues

by site-directed mutagenesis

Functional residues identified by in silico approaches were

further validated by site-directed mutagenesis where

Tyr327 and His368 of Ptc-GT mutated by Asn327 and

P.torridus --------------------------------------------MYMNYAVASSHPLSTF 16 T.acidophilum ------------------------------------MFRSRPNALSQRSVIASSSELASL 24 G.thermonitrificans ---------------------------MDYLYHP--YPSQRMTVFAKNGIVATSQPLAAQ 31 B.halodurans -----------------------MSVMFDPQSYP--YPSRRNVVYAKNGMVATSQPLAAQ 35 D.radiodurans --------------------------MLRPMTHNPEYPVVRRPAYARRGMVATSQPLAAQ 34 T.thermophilus ---------------------------MDLTYYP--YPSRRHVVLGRRGAVATSQPLAAL 31 E.coli MIKPTFLRRVAIAALLSGSCFSAAAAPPAPPVSYGVEEDVFHPVRAKQGMVASVDATATQ 60 . :*: :: P.torridus VGNEILKDGGNAYDAAIATSAALVVVQPHLNGLGGDFFSTIIDN--DIYSINGSGNAPEL 74 T.acidophilum AGRDILKRGGNIFDAALAVSAMLCVTQNNLCGLGGDLFALIRDENGQIMDLNGSGQASRA 84 G.thermonitrificans AGLEVLKKGGNAIDAAIATAACLTVVEPTSNGIGGDAFALVWTNG-KLYGLNASGYAPAA 90 B.halodurans AGLDILKAGGNAIDAAIATATALTVLEPTSNGIGSDAFALVWTKG-KLHGLNGSGRAPMS 94 D.radiodurans AGLSILQAGGNAVDAAVATAAALTVVEPTSNGLGGDAFALVWAGG-ELHGLNASGAAPAA 93 T.thermophilus AGMEVLLKGGSAVDAAIAMAACLTVVEPTSNGIGGDLFALVWDG--TLHGLNASGKSPMA 89 E.coli VGVDILKEGGNAVDAAVAVGYALAVTHPQAGNLGGGGFMLIRSKNGNTTAIDFREMAPAK 120 .* .:* **. ***:* . * * . .:*.. * : :: :. P.torridus ATIEFFHRNGYNKIPEQGPLSSFS--IPGLVSSWEILYKN-ATMKLEKLFSRAISFAMDG 131 T.acidophilum VSIDYYESMGLTKIPERGPYAAIT--VPGIAGSWDEIFRKFATMDIADILEPAIRTASAG 142 G.thermonitrificans ISLDVLKERGYT-EMPKYGFAPVT--VPGAPAAWAALSKRFGRLSLAETLAPAIAYAENG 147 B.halodurans LTMEAVKAKGYEQELPPYGVIPVT--VPGAPGAWAELAKMYGNLPLAASLAPAIRYAEEG 152 D.radiodurans LSLEALP----GGEMPKYGWTPVT--VPGAVRGWTDLHGRFGRLDFAQVLAPAIRYAREG 147 T.thermophilus LTPERLP-----GRMPERGWLPVT--VPGAVSGWRALHERWGRLPFAEVLAPAIRYAEEG 142 E.coli ATRDMFLDDQGNPDSKKSLTSHLASGTPGTVAGFSLALDKYGTMPLNKVVQPAFKLARDG 180 : : .: ** .: . : : . *: * * P.torridus FIPSNSILKAIKN---FKYGDVDFN------NIYYNNE------RLLVQRALGKTLKLLA 176 T.acidophilum FPITQNYSDSIARSAPVIGQYRGWS------SIFMPNGSVPVAGEILKQPDLAESFRLMS 196 G.thermonitrificans YPVSPVLGKYWANAYRVYKEALHGPEFGSWFETFAPAGRAPNIGEVWASPDHAATLRSIA 207 B.halodurans YPVTPTLAKYWKAAYDRVKTEWTDDVYQPWFDTFAPKGRAPRVGEVWRSQGHADTLRSIA 212 D.radiodurans YPLSPVLAANWARAIRSY-WALNLPIFEDWFRTFAPDGFTPRPGALWRSEGHARTLELIA 206 T.thermophilus FPVGPETARSWRRAEGVF-LPLEGPEFGPFKEVFFPGGRAPRAGEVWRSPLHAKTLREIA 201 E.coli FIVNDALADDLKTYGSEVLPNHENSK-----AIFWKEGEPLKKGDTLVQANLAKSLEMIA 235 : : . . ::. :: P.torridus EKGLESFYHGDIARAIEDDMIKKHGLIRFNDLDSYSASVVKPLEIRYRNYSVYTNPP-VS 235 T.acidophilum EEGFRSFYDGSLADIIIAGLEGTGSPLSDRDLRVYRPLIGKPVFTDLDEFRIYETSP-NS 255 G.thermonitrificans ETEAESFYRGELAEKIVAFSKQYNGFLTLEDLAEYEPEWVEPISVSYHGYDVWEIPP-NG 266 B.halodurans ESNGESFYRGELADQIHAFFDKHGGYLTKEDLACYRPEWVEPISIDYRGYRVWEIPP-NG 271 D.radiodurans QTGGAAFYEGELAGQIDAHAQATGGLLRGSDLAAHRSEWVKPIHTDWLGHRVYEIPP-NG 265 T.thermophilus ESYGESLYRGALAEALLRFSEATGGLLTREDLEAHAPEWVTPLSTEYKGLTVWELPP-NG 260 E.coli ENGPDEFYKGTIAEQIAQEMQKNGGLITKEDLAAYKAVERTPISGDYRGYQVYSMPPPSS 295 : :* * :* : . : ** : . *: :: .* . P.torridus QGATALYWLNRLNKYDLYT--MPDDLYYSSLINEMYPSYEFRKSMIYDGSS-ISNDDLLN 292 T.acidophilum QGITVIEWIRGMESHGYDSR-TMWEAKIEDIFETMEEAYDKRRKITDPSYMNIAQHDSAN 314 G.thermonitrificans QGLVALMALNIMNGFDVPNV--PDVETYHRQIEAMKLAFADGKAYIADRRYMNYRVDELL 324 B.halodurans QGLVALEALNIVKGFEFYHK--DTVDTYHKQIEAMKLAFVDGMKYVTEPSDMSVSVEQLL 329 D.radiodurans QGIAALVALNVLGAERLPEL-RDDPAGLHLQIEAMKRGFHDAHSYVADPRHVPVDVEGLL 324 T.thermophilus QGVAVLLALNLLEGFDLRPE---DPWSYHVQIEAMRLALADTYRFVADPRHMELAPEAFL 317 E.coli GGIHIVQILNILENFDMKKYGFGSADAMQIMAEAEKYAYADRSEYLGDPDFVKVPWQALT 355 * : :. : : . : P.torridus NYSYSK-------NKKDE-------------KYSDTTAFSVFDGDAG-ISAIQSNYMGFG 331 T.acidophilum GKKDGL-------PKRDHN------------DIGDTTYFSISDSEGRSVSIIQSNYMGFG 355 G.thermonitrificans SESFAA-------MRRAQIGTEALTPEPGTPPKGGTVYLAAADGEGNMVSFIQSNYMGFG 377 B.halodurans SDEYAT-------ERRKEIGEQALTPEPGTPPRGGTVYLATADGDGNMVSFIQSNYMGFG 382 D.radiodurans SAANAE-------RHRALLGEQAHDPATHAPSTGGTVYLAAADDEGGMVSMIQSNYMGFG 377 T.thermophilus SKAYAA-------ERRRLIGERALPRVLPGLRPEGTVYLAAADG-EVMVSLIQSNYQGFG 369 E.coli NKAYAKSIADQIDINKAKPSSEIRPGKLAPYESNQTTHYSVVDKDGNAVAVTYTLNTTFG 415 . . .: *. : * :: : ** P.torridus SGHSIHGYGINMNNRGSYFTLDKNHK----------NALKPGKKTFHTLMS-TILN--GD 378 T.acidophilum SGIVPKGTGFVLQNRGSYFTLQRDHP----------NALMPGKRTFHTLAA-CMVEKEHD 404 G.thermonitrificans SGLVVPGTGIALHNRGHNFVFDENHP----------NGLAPRKKPYHTIIPGFLTKGG-K 426 B.halodurans SGVVVPGTGIAMQNRGHNFSLDPNHD----------NALKPGKRTYHTIIPGFLTKND-Q 431 D.radiodurans SGVVVPGTGIALHNRGHNFHTDPAHP----------NALAPGKRPYHTIIPGFLGRADGT 427 T.thermophilus SGVLVPGTGIALQNRGLGFSLEEGHP----------NRVGPGKRPFHTIIPGFLAREG-K 418 E.coli TGIVAGESGILLNNQMDDFSAKPGVPNVYGLVGGDANAVGPNKRPLSSMSPTIVVKDG-K 474 :* *: ::*: * . * : * *:. :: . : . P.torridus KLILLGSMGGDIQPQVNVQIITRIIDLNYNIQDAISYPRFAYPASIYG--------DADL 430 T.acidophilum LYASLGSMGGDIQPQVQMQILMEILKDNTDPQAILDKPRWTEPYTIYEAPGAVYVESEEL 464 G.thermonitrificans PIGPFGVMGGFMQPQGHMQVIMNTVDFALNPQAALDAPRWQWMEGKTVLVEP---HFPRH 483 B.halodurans PIGPFGVMGGFMQPQGHMQVMMNTIDFGLNPQAALDAPRWQWTNGKQVQVEP---TFPVD 488 D.radiodurans PVGPFGVMGGFMQPQGQLQVVVNTVRYGMNPQQALDAPRWQWLQGRTVEVEP---ALGDQ 484 T.thermophilus PLGPFGVMGGFMQPQGHVQVVVGLADFGLNPQAALDRPRWQVVPGDEVLLEP---GIPQA 475 E.coli TWLVTGSPGGSRIITTVLQMVVNSIDYGLNVAEATNAPRFHHQWLPDELRVEKG--FSPD 532 * ** :*:: : . **: P.torridus YS--EKGIVPGSKYIDGLSSMMGHAQG--ILIEDDVHAGFDPRGDGLLKYHL------- 478 T.acidophilum YRNVSKQISGRKVVLRDVSQEFGTAQITTLIRGDVVVGAADPRGDGIAIPYS------- 516 G.thermonitrificans IAEALARKGHDICVALDGGPFGRGQIIWRDPDTGVLAAGTEPRTDGAVAAW-------- 534 B.halodurans IAQALVRRGHKIQVVLDEGAFGRGQIIWRDPTTGVLAGGTEPRTDGQVAAWEGHHHHHH 547 D.radiodurans LARALVARGHDVRVQLDPGSFGRGQMIRRDPDTGVLEGGTESRTDGHIALW-------- 535 T.thermophilus TALFLKDLGHRVRYEAEYGLFGRGQVVFR--LGEALVGASDPRAEGLALAW-------- 524 E.coli TLKLLEAKGQKVALKEAMG----STQSIMVGPDGELYGASDPRSVDDLTAGY------- 580

Fig. 5 Multiple sequence

alignment of Ptc-GT with other

extremophiles and thermophiles

c-GTs. Ptc-GT was aligned

with T. acidophilum,

G. thermonitrificans,

B. halodurans, D. radiodurans,

T. thermophilus and E. colic-GTs by ClustalW. The

conserved residues Thr (T308 in

Ptc-GT) crucial for

autoprocessing are highlighted

in red. Oxyanion hole residues

Gly, Gly (G387, G388 in

Ptc-GT) conserved among all

c-GTs are highlighted in green.

Other active site residues which

are conserved or semi-

conserved (S68, S325, Y32,

R346, Y349, F367, H368, T369,

M386 and Q391 in Ptc-GT)

among all c-GTs are highlighted

in cyan. Lid loop extending

toward the active site (from

P438 to G449 of Ecc-GT) and

absent in Ptc-GT and other

c-GTs are underlined and

highlighted in magenta. Signal

peptide in Ecc-GT is underlinedand highlighted in dark(Castellano et al. 2010, 2011;

Futterer et al. 2004; Ruepp et al.

2000; Takami et al. 2000;

Suzuki et al. 1986) (color figure

online)

36 Extremophiles (2013) 17:29–41

123

Ser368 to that of E. coli introduced significant transpepti-

dase activity in both the mutants. The catalytic efficiency

was 10.94 ± 1.0 min-1 lM-1 and 10.37 ± 1.5 min-1 lM-1

for mutants, Y327N and H368N, respectively (Table 3).

However, both mutations resulted in lowering of Km and

declined in hydrolytic rate. Comparative docking analysis of

mutant Y327N and wild type with acceptor glycyl-glycine

revealed that the ligand docked perfectly in binding pocket of

mutant where five residues (L87, E90, Y305, N327, S348)

hydrogen bonded with glycyl-glycine, in contrast to wild type,

where the acceptor did not dock perfectly in binding pocket

and only four residues (S348, F350, T351, Y425) were

hydrogen bonded (Fig. 7a, b), and the catalytic nucleophile

(T308) was found to be away from ligand binding core. This

is further strengthened by comparative fluorescence emission

analysis where a fluorescence shift from 330 nm of mutant

Y327N without acceptor to 320 nm in case of DOPA and

345 nm in the presence of glycyl-glycine and ethylamine,

respectively, was observed while only quenching was

observed in case of wild type in the presence of acceptors

(Fig. 7c, d).

Thermostability of Ptc-GT

To analyze the molecular features responsible for adapta-

tion to extreme condition in c-GTs, some parameters

related to amino acid composition (Table 1) and structure

(Table 2) of Ptc-GT were analyzed and compared in detail

with a mesophilic, Ecc-GT and a thermophilic, Gtc-GT.

Compositional amino acid analysis of Ptc-GT revealed

that, when compared with Ecc-GT and Gtc-GT, the protein

has higher uncharged polar residues Gln, Asn, Thr and Ser,

coupled with lower Arg/Lys ratio than Gtc-GT but higher

than Ecc-GT (Table 2). Larger content of arginine favors

protein thermostability whereas larger content of lysine

decreases protein thermostability, which is in accordance

with Szilagyi and Zavodsky (2000). Ptc-GT amino acid

sequence contains higher content of charged residues Glu,

Asp, Lys and Arg as compared to Ecc-GT and Gtc-GT; this

is in support of Ptc-GT thermostability (Haney et al. 1999).

To identify structural basis for thermostability of

Ptc-GT, detailed structural features of all three c-GTs were

analyzed (Table 3). Ptc-GT showed higher total accessible

surface area, mean residue accessible surface area and

mean residue volume with respect to Ecc-GT and Gtc-GT.

Ptc-GT also showed higher molecular mass and volume

similar to Ecc-GT and Gtc-GT (De Vendittis et al. 2008).

Discussion

Extremophiles are an unlimited reservoir of enzymes with

potential biotechnological properties, since extremophilic

conditions lower the risk of contamination in addition to

better solubility of substrates (Burg 2003). Among thermo-

philes and extremophiles, archaea are relatively a less

explored group due to extreme culture conditions and slow

growth rate. However, tapping these organisms has now

become possible due to growing whole genome database.

Among these, Picrophilus torridus, a thermo-acidophilic

archaeon has gained attention in the recent times because of

its ability to live under highly acidic, at pH values close to pH

0.0 (Angelov et al. 2006). Previously few enzymes namely

pantothenate kinase, a-mannosidase and a-glucosidase,

2-keto-3-deoxygluconate-specific aldolase, glucose/galactose

dehydrogenase and glucoamylase have been described in

P. torridus (Takagi et al. 2010; Angelov et al. 2005; Reher

et al. 2010; Angelov et al. 2006; Schepers et al. 2006).

Fig. 6 Three-dimensional representation of monomeric form and

binding pocket. a The monomeric form of modeled Ptc-GT structure

where b-sheets were sandwiched by a-helices. Large subunit com-

ponent a-helices showed in green, cyan and lemon color and b-sheets

showed in blue and green colors; whereas small subunit component

a-helices showed in brown and b-sheets in red color. b Binding mode

of c-glutamyl moiety (dotted form) inside the active site of Ptc-GT

(color figure online)

Extremophiles (2013) 17:29–41 37

123

The gene, ptc-gt from P. torridus was cloned and

expressed in pET 51b-E. coli Rosetta cells using IPTG

induction. However, the enzyme was mostly recovered as

pro-c-GT in an inactive form. The autoprocessing of the

enzyme was poor but was enhanced to nearly 10-fold when

recombinant E. coli was grown in the presence of

n-hexadecane. This is in confirmation with the earlier

reports where use of oxygen vectors has been suggested to

improve the yield of recombinant proteins by improving

oxygen transfer rate (Giridhar and Srivastava 2000). c-GT

of P. torridus was found to be heterodimeric protein

composed of two subunits with apparent molecular mass of

30 and 17 kDa, respectively, which is in confirmation with

other c-GTs, encoded by a single gene and processed into

two subunits, large and small (Boanca et al. 2006; Suzuki

and Kumagai 2002; Kinlough et al. 2005).

c-GT was purified using nickel affinity chromatography

with a recovery of 61 % and studied biochemically.

Ptc-GT displayed only hydrolase activity which is in

agreement with other thermophiles and extremophiles such

as T. thermophilus and D. radiodurans, and G. thermo-

denitrificans which all are reported to be hydrolytic

(Castellano et al. 2010, 2011). More than 50 % of enzymes

were active over a broad pH range from 4.0 to 8.0 and

optima at pH 7.0. Generally, c-GTs from various organisms

are reported to be maximally active at pH 8.0 with slight

activity in acidic pH range (Tate and Meister 1985; Wang

et al. 2008). The enzyme was acid stable and has[50 % of

activity even at pH 2.0 in contrast to earlier reports where

c-GTs are known to be stable between neutral and alkaline

range (Tate and Meister 1985; Wang et al. 2008). This

property may be attributed to the differences in amino acid

Wavelength (nm)290 30

0310 32

0330 340 35

036

037

038

0390 400 41

042

043

044

0450 460

Flu

ores

cenc

e In

tens

ity (

au)

0

100

200

300

400

500

600

700

800

900

1000

Y327N

Y327N+Ethylamine

Y327N+L-DOPA

Y327N+Glycylglycine

Wavelength (nm)29

030

031

032

033

034

035

036

037

038

039

040

041

042

0430 44

045

046

0

Flu

ores

cenc

e In

tens

ity (

au)

0

50

100

150

200

250

300

350

400

Ptγ – GT

Ptγ – GT+Ethylamine

Ptγ – GT+L-DOPA

Ptγ – GT+Glycylglycine

a b

c d

Fig. 7 a, b Binding mode of glycyl-glycine with mutant (Y327N)

and wild. a Glycyl-glycine strongly held up by five residues (L87,

E90, Y305, N327, S348) where N327 of mutant makes two hydrogen

bonds with glycyl-glycine. b Binding mode of glycyl-glycine

with wild enzyme where it was weakly held up by four residues

(S348, F350, T351, Y425) where nucleophile (T308) is found to be

far away from ligand binding core. c, d Comparative fluorescence

emission spectrum with acceptors glycylglycine, ethyl amine and

DOPA of c mutant d wild types

38 Extremophiles (2013) 17:29–41

123

composition which would have been due to evolutionary

selection due to the acidic habitat of P. torridus. The

enzyme exhibited[50 % of activity between 40 and 60 �C

with optima at 60 �C similar to other c-GTs from meso-

philes and extremophiles (Suzuki and Kumagai 2002; Abe

et al. 1995). However, it was highly thermostable with a

t1/2 of 1 h at 50 �C and 30 min at 60 �C and retained

100 % activity even after 24 h at 45 �C which is similar to

G. thermodenitrificans which retained up to 80 % activity

under same conditions (Castellano et al. 2010). However,

c-GT from E. coli and Bacillus were comparatively ther-

molabile where c-GTs were inactivated at [45 �C within

15 min–1 h (Suzuki and Kumagai 2002; Abe et al. 1995).

The amino acid sequence and structural features which are

known to govern thermostability were found to fit well

with experimentally observed thermostability in Ptc-GT

(Szilagyi and Zavodsky 2000; Haney et al. 1999; De

Vendittis et al. 2008). It showed high arginine and charged

amino acid content along with larger molecular mass and

protein volume similar to that of G. thermodenitrificans

(Castellano et al. 2010).

The catalytic properties suggested that the present

enzyme was different from E. coli as it did not show

transpeptidation. The position-specific multiple sequence

alignment and structural analysis suggested that only two

residues Gly386 and Gly387 were conserved in Ptc-GT and

all the rest binding sites were different. However, all these

residues of Ptc-GT were conserved and semi-conserved in

extremophiles. This suggests that the differences in binding

residues of extremophiles may be the key reason of

extremophilic c-GTs being only hydrolytic in contrast to

E. coli. Therefore, these residues were mutated to that of

E. coli. Tyr327 and His368 of Ptc-GT mutated by Asn327

and Ser368, respectively, introduced significant transpep-

tidase activity in both the mutants. None of the other

mutations could introduce transpeptidase activity. How-

ever, in all the mutations, the hydrolysis rate declined with

lowering of Km, indicating the improved substrate binding

efficiency after mutations. This is well supported by

comparative docking analysis of mutant and wild protein

with glycyl-glycine as acceptor substrate. The acceptor was

observed to be held by six hydrogen bonds in Y327N

mutant in contrast to the wild, where the acceptor was not

held properly in the binding pocket. This indicates that the

transpeptidase activity in case of mutant is due to the

change in protein conformation resulting in better acceptor

binding. This is further strengthened by fluorescence shift

of mutant, Y327N in the presence of acceptors like glycyl-

glycine, DOPA and ethylamine while no such shift was

observed in case wild protein. Mutation studies suggest that

the differences lie in acceptor site of pocket, and due to

Table 2 Structural parameters of P. torridus compared with E. coliand G. thermodenitrificans

Structural parameters Ptc-GT Ecc-GT Gtc-GT

Total accessible surface area

(ASA, A2)

38,510 37,745.4 38,449

Accessible surface area of

backbone (A2)

4,798.2 4,935.6 4,354

Accessible surface area of side

chain (A2)

33,711.8 32,810 34,095

Mean residue ASA (A2) 45.1 34.4 36.3

Exposed non polar ASA (A2) 21,949 22,343 22,040

Exposed polar ASA (A2) 8,745.8 7,861.6 7,591.6

Exposed charged ASA (A2) 7,815.2 7,541 8,817.2

Fraction non polar ASA (%) 57 59 57

Fraction polar ASA (%) 23 21 20

Fraction charged ASA (%) 20 20 23

Total volume (A3) 128,518.8 144,928.8 139,736.8

Mean residues volume (A3) 150.5 132 132.1

b-Sheet content (%) 26 24 27

a-Helix content (%) 30 36 35

Coil content (%) 42 38 37

Fraction residues with hydrogen

bond (%)

70 78 77

Table 3 Steady state kinetics of Ptc-GT versus mutants

Protein Hydrolytic activity (without acceptor) Transpeptidase activity (with acceptor)

Km (lM) Vmax

(lmoles-1 ml-1 min-1)

Kcat/Km

(min-1 lM-1)

Km (lM) Vmax

(lmoles-1 ml-1 min-1)

Kcat/Km

(min-1 lM-1)

Ptc-GT 100 ± 1.2 5.0 ± 1.8 2.36 ± 1.1 – – –

Y327N 66.6 ± 1.0 1.6 ± 1.6 1.50 ± 1.3 25.0 ± 0.6 5.8 ± 0.4 10.94 ± 1.0

H368S 14.3 ± 0.9 2.0 ± 1.0 6.06 ± 2.0 25.0 ± 1.3 5.5 ± 0.5 10.37 ± 1.5

Y327E 66.6 ± 1.0 3.3 ± 1.6 3.60 ± 1.7 – – –

R346A 28.6 ± 1.4 2.2 ± 1.2 3.26 ± 1.0 – – –

R346E 66.0 ± 0.5 4.0 ± 1.0 2.86 ± 1.4 – – –

Y349D 66.6 ± 1.2 1.3 ± 0.9 2.34 ± 1.4 – – –

Extremophiles (2013) 17:29–41 39

123

these differences, Ptc-GT has lost transpeptidase activity as

evident by the recovery of transpeptidase activity on

mutations, Y327N, and H368S. Phylogenetic analysis also

revealed three clusters of extremophiles, mesophiles and

eukaryotes, again supporting the evolutionary divergence

of extremophiles.

Conclusion

In the present study, c-glutamyl transpeptidase of a thermo-

acidophilic archaeon Picrophilus torridus was cloned and

expressed using E. coli Rosetta-pET 51b(?) expression

system. The enzyme was expressed at 37 �C/200 rpm after

3 h of IPTG induction. It was improved nearby 10-fold in

the presence of 2 % hexadecane. The recombinant protein

was synthesized as a precursor heterodimeric protein of

47 kDa with two subunits of 30 kDa and 17 kDa, respec-

tively. The enzyme was characterized biochemically and

found to be acid and thermo stable at a significant level. In

silico approaches showed a lot of differences in binding

residues of Ptc-GT with respect to Ecc-GT except Thr308

as autoprocessing and enzymatic residues, and Gly387 and

Gly388 as oxyanion hole residues. Ptc-GT residues were

found to be conserved or semi-conserved in other

extremophiles c-GTs which was also hydrolytic in nature.

Mutation at binding pocket residue, i.e., Tyr327 mutated to

Asn327 to that of E. coli introduced significant transpep-

tidase activity which was well supported by comparative

docking and fluorescence analysis spectra of mutant and

wild with acceptor substrates.

Acknowledgments Financial assistance from DU/DST-PURSE

grant and Misc. R & D Grant, University of Delhi is acknowledged.

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