Microbiology Papers in Press. Published February 8, 2015 as doi:10.1099/mic.0.000052

Microbiology A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to maintain cell-shape and intrinsic beta-lactam resistance --Manuscript Draft-Manuscript Number:

MIC-D-14-00066R2

Full Title:

A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to maintain cell-shape and intrinsic beta-lactam resistance

Short Title:

Involvement of Leu182 in the physiology of E. coli PBP5

Article Type:

Standard

Section/Category:

Physiology and metabolism

Corresponding Author:

Anindya S Ghosh Indian Institute of Technology Kharagpur Kharagpur, WB INDIA

First Author:

Mouparna Dutta

Order of Authors:

Mouparna Dutta Debasish Kar, M. Tech. Ankita Bansal Sandeep Chakraborty Anindya S Ghosh

Abstract:

PBP5, a DD-carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance in E. coli. A strain lacking PBP5 loses intrinsic beta-lactam resistance and simultaneous lack of two other PBPs results in aberrantly shaped cells. PBP5 expression in trans complements the shape and restores the lost beta-lactam resistance. PBP5 has a 'Ω loop' like region similar to that in Class A beta-lactamases. It was previously predicted that Leu182 present in the 'Ω -like' loop of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and biochemical effects of the Leu182Glu mutation in PBP5. Upon the over-expression in septuple PBP mutants, ~75% cells were abnormally shaped and intrinsic beta-lactam resistance maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5) retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and peptidoglycan mimetic substrates were ~10 times lesser than that of wild type. Superimposition of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key residues is a possible reason for the low turnover numbers. Therefore, we establish the involvement of Leu182 in maintaining the physiological and biochemical behavior of E. coli PBP5.

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1

Title:

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A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to

3

maintain cell-shape and intrinsic beta-lactam resistance

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Short Title: Involvement of Leu182 in the physiology of E. coli PBP5

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Authors: Mouparna Dutta, Debasish Kar, Ankita Bansal, Sandeep Chakraborty1,2, Anindya S.

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Ghosh*

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Affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur,

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Kharagpur-721302, India

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1

Plant Sciences Department, University of California, Davis, 95616, USA

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2

Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India

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*Corresponding Author:

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Anindya S. Ghosh, E-mail: [email protected]; Tel: +91-3222-283798

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(i) Summary: 191 words

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(ii) Main text: 4165 words

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Tables: 04

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Figures: 04

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Supplementary Material: 01 (contains 02 figures and 01 table)

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Summary:

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PBP5, a DD-carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance

28

in E. coli. A strain lacking PBP5 loses intrinsic beta-lactam resistance and simultaneous lack of

29

two other PBPs results in aberrantly shaped cells. PBP5 expression in trans complements the

30

shape and restores the lost beta-lactam resistance. PBP5 has a ‘Ω loop’ like region similar to that

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in Class A beta-lactamases. It was previously predicted that Leu182 present in the ‘Ω –like’ loop

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of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and

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biochemical effects of the Leu182Glu mutation in PBP5. Upon the over-expression in septuple

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PBP mutants, ~75% cells were abnormally shaped and intrinsic beta-lactam resistance

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maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5)

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retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and

37

peptidoglycan mimetic substrates were ~10 times lesser than that of wild type. Superimposition

38

of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key

39

residues is a possible reason for the low turnover numbers.

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involvement of Leu182 in maintaining the physiological and biochemical behavior of E. coli

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PBP5.

Therefore, we establish the

42 43

Keywords: E. coli; penicillin-binding protein 5; omega loop; cell-shape; site-directed

44

mutagenesis; DD-CPase

45 46

47 48 49

1.0. Introduction

50

One of the main bacterial cell wall components is the peptidoglycan layer, which is

51

indispensible for cellular viability and maintenance of cellular morphology. Penicillin-binding

52

proteins (PBPs) are the enzymes required for the cross-linking of the glycan chain

53

(transglycosylase) and peptide chain (transpeptidase). Majority of these proteins have three

54

signature motifs, namely, SXXK, SXN and KTG, and these motifs are responsible for their

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respective enzymatic activities (Ghuysen, 1991). In E. coli, five PBPs (PBP4, 5, 6, DacD and

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AmpH) are documented as DD-Carboxypeptidase (DD-CPase) (Korat et al., 1991; Holtje, 1998;

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Nelson and Young 200 and 2001; Baquero et al., 1996 and Gonzalez-Leiza et al., 2011). These

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PBPs are thought to prevent the unwanted crosslink formation by removing the terminal D-

59

alanine residue from the pentapeptide side-chain of N-acetyl muramic acid (Holtje, 1998 and

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Ghosh et al., 2008). PBP5 is the key DD-CPase that helps maintain a uniform cell-shape in E.

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coli (Nelson et al, 2000, 2001 and Ghosh et al., 2008). E. coli mutants lacking at least three DD-

62

CPases including PBP5 show aberrant morphology. However, the abnormalities are abolished

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upon ectopic expression of PBP5 (Nelson and Young, 2000; Ghosh and Young, 2003). The

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ability of PBP5 to repair the cellular oddities depends on a stretch of 20 amino acids (200-219)

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around the KTG motif of PBP5, known as the ‘morphology maintenance domain’ (MMD)

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(Ghosh and Young, 2003). Apart from maintaining the cell shape, PBP5 also plays an important

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role in maintaining the intrinsic beta-lactam resistance in E. coli (Sarkar et al., 2010). The

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absence of PBP5 sensitizes the E. coli cell to beta-lactam antibiotics, while in trans expression of

69

the same helps to reverse the lost resistance (Sarkar et al., 2010). It is speculated that the high

70

copy number of PBP5 at the logarithmic phase of growth may be responsible for trapping the

71

beta-lactam antibiotics, which prevents them from binding to the essential PBPs (Sarkar et al.,

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2010; Sarkar et al., 2011).

73

Several mutation analyses have been studied for assessing the activity of PBP5 in vitro.

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The PBP5 ‘74-90 loop’ deletion mutant, Gly105Asp mutant and the modified Cys115 resulted in

75

the loss of DD-CPase activity individually due to inefficient deacylation (Nicholas et al., 2003;

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Nicola et al., 2005). The particular defects in deacylation reveal the intimate role of SXN motif

77

especially Ser110 in the catalytic mechanism (Nicola et al., 2005). In addition to the active-site

78

residues, Asp175 also serves as a determinant of in vitro DD-CPase activity, but not for the

79

penicillin-binding activity (van der Linden et al., 1994).

80

It is speculated that PBP5 may have beta-lactamase like activity as it shares a common

81

ancestry with beta-lactamases (Zhang et al., 2007). Nevertheless, PBP5 in its soluble or in

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membrane-bound form does not show beta-lactamase activity under physiological pH condition

83

(Sarkar et al., 2010), thoughPBP5 has a ‘Ω-loop’ like region (a characteristic for the beta-

84

lactamases) similar to that of TEM-1 beta-lactamases (Zhang et al., 2007; Ghosh et al., 2008).

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The orientation of His151 in this loop is similar to that of Glu166 in TEM-1, which might help in

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accessing water molecule during deacylation of beta-lactams by PBP5 (Davies et al., 2001;

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Ghosh et al., 2008). It has been reported that the soluble form of PBP-A of

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Thermosynechococcus elongatus possesses 50-fold increase in penicillin hydrolysis upon L158E

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mutation at the ‘Ω-loop’ like region, but its physiological effect is yet unknown (Urbach et al.,

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2008). Recently, based on in silico predictions, it is hypothesized that the Leu153 of ‘Ω-loop’

91

like region in E. coli PBP5 could be the best candidate for mimicking Glu166 residue of Class A

92

beta-lactamases (Chakraborty, 2012).

93

In the present study, we investigated whether the alteration in the 'Ω-loop' could affect the

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physiological function of PBP5. Accordingly, the leucine present in the 'Ω-loop’ of both

95

membrane-bound (at 182nd position) and the soluble (at 153rd position) constructs of E. coli

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PBP5 are replaced with glutamic acid residue. Physiological functions of the mutant in terms of

97

cell shape and intrinsic beta-lactam resistance are assessed. Subsequently, the alterations in

98

functions of the mutant are verified through in vitro biochemical analyses and finally the possible

99

reason for the altered functions are explained by using in silico molecular modeling and

100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115

superimposition studies.

116

2.0. Materials and Methods

117

2.1. Bacterial strains, reagents and growth conditions

118

E. coli K12 (CS109) strain and its PBP deletion mutants, CS703-1 (∆PBP1, 4, 5, 6, 7,

119

AmpC and AmpH) and AM15-1(∆PBP5) were used for this study (Nelson et al., 2002 and

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Sarkar et al., 2010) (Table. 1). The strains were grown in Luria-Bertani (LB) broth and Muller-

121

Hinton (MH) broth (HiMedia, Mumbai, India). Chloramphenicol (20 µg ml-1) or kanamycin (50

122

µg ml-1) was added wherever necessary. Unless otherwise specified all the chemical and reagents

123

were purchased from Sigma-Aldrich (St. Louis, MO, USA).

124 125

2.2. Construction of the clones for full-length PBP5L182E and soluble form of the protein

126

(smPBP5)

127

The clone for PBP5L182E was created on the membrane-bound form and smPBP5 were

128

created on soluble form of PBP5 by using commercially available site-directed mutagenesis kit

129

(Stratagene Co., La Jolla, Calif.) to generate the plasmids pPJ5L182E and pTS5LE, respectively

130

(Table. 1). The plasmid, pPJ5 was used as template to make pPJ5L182E, while pT7cPBP5 was

131

used as template to make pTS5LE. Same set of oligonucleotide primer pairs were used for each

132

reaction.

133

ctgaccatcagcatcctcaccatgtaccgtctgg-3'. The gene for both sPBP5 and smPBP5 were sub-cloned

134

from pT7-7K to pET28a (+) at the sites of NdeI and HindIII to generate pET5 and pET5LE,

135

respectively (Table. 1). Finally, the mutations in the respective clones were confirmed by

136

sequencing through commercial sequencing service (MWG Biotech Inc, Bangalore, India).

Those

were

137 138

2.3. Assessment of Cell shape

5'-ccagacggtacatggtgaggatgctgatggtcag-3'

and

5'-

139

The E. coli strain CS703-1 containing pPJ5L182E was expressed by inducing with

140

0.005% arabinose at the early log phase as previously described (Nelson and Young, 2001).

141

Cells were then incubated for 2-4 h and treated with aztreonam at the final concentration of 5 µg

142

ml-1 (Nilsen et al, 2004). Further, cells were washed with 1X PBS (pH 7.4) and cell morphology

143

was checked by fixing the cells onto poly-lysine coated glass slides and subsequently visualized

144

the live cells under the OLYMPUS IX 71 phase-contrast microscope (Olympus Inc., Japan).

145 146

2.4. Determination of Minimum Inhibitory Concentration (MIC)

147

MIC values were determined by the microtiter plate dilution method as described earlier

148

(Sarkar et al., 2011). Total volume of MH broth in each well was made up to 300 µl by adding

149

overnight grown culture of the respective E. coli strains at a concentration ~105 cells per well.

150

The antibiotics were tested by two-fold step dilution (Sarkar et al., 2010) and finally, the

151

microtiter plates were incubated at 37º C for 18 h. Bacterial growth was monitored by measuring

152

the optical density at 600 nm using a Multiskan Spectrum spectrophotometer (model 1500;

153

Thermo Scientific, Nyon, Switzerland). MIC values were tested in triplicate and repeated at least

154

six times for accuracy.

155 156

2.5. Expression and purification of the soluble form of wild type PBP5 (sPBP5) and its mutant

157

(smPBP5)

158

The expression of sPBP5 and smPBP5 were carried out in E. coli BL21 star under the

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control of T7 promoter. The bulk expression was obtained in cytoplasmic fraction by growing

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the cells at 30º C for 8 h incubation in the presence of 0.05 mM isopropyl-β-

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thiogalactopyranoside (IPTG). The N-terminal His-tag containing sPBP5 and smPBP5 were

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purified using Ni-NTA affinity chromatography. The proteins were eluted from the affinity

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column in the elution buffers containing 50 mM and 100 mM of imidazole in a buffer (10 mM

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Tris-HCl, 300 mM NaCl buffer, pH 8.0) at 4° C. The eluted fractions were subjected to 12%

165

SDS-PAGE to determine their molecular weights and their activity was assessed prior to

166

collection. The fractions collected were dialyzed for 12 h with three changes of same buffer

167

without imidazole and was further concentrated by using Amicon Ultra-4 Devices (Millipore,

168

Billerica, MA).

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2.6. Fluorescent penicillin binding assay

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The stability and activity of the PBPs were checked by labeling with Bocillin-FL

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(Invitrogen, Carlsbad, CA), a fluorescent penicillin. The membrane bound PBPs (~300 µg) and

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the soluble PBPs (~25 µg) were incubated at 35º C for 30 min with 50 µM and 100 µM of

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Bocillin-FL, respectively (Zhao et al., 1999; Chowdhury et al., 2010). Further, the proteins were

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analyzed through 12% SDS-PAGE and visualized in Typhoon FLA 7000 (GE Healthcare Bio-

176

Sciences AB, Uppsala, Sweden).

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2.7. Kinetics analysis of smPBP5 with penta-peptide substrate

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The DD-CPase activity exerted by both sPBP5 and smPBP5 were checked for the

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artificial substrate, Nα,Nє-diacetyl-Lys-D-Ala-D-Ala (AcLAA) and peptidoglycan mimetic

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pentapeptide substrate, L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala (AGLAA) (USV custom peptide

182

synthesis, Mumbai, India) in 10 mM Tris-HCl buffer (pH 8.0) (Chowdhury et al.,2010). The

183

reactions were carried out in 60 µl for each soluble PBPs where 2 mg of the each proteins was

184

mixed with the respective peptides at a concentration range of 0.25–12 mM. The remaining

185

volume was adjusted with 50 mM Tris-HCl, pH 8.5. The reaction mixture was incubated at 37

186

°C for 30 min and after which freshly prepared enzyme–coenzyme mixture (140 µl) was added.

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The enzyme-co-enzyme mixture was prepared by mixing 50 mM Tris-HCl, pH 8.5; 0.3 mg ml-1

188

FAD; 10 mg ml-1 horseradish peroxidase; and 5 mg ml-1 D-amino acid oxidase at a ration 20: 10:

189

5: 1). This resultant mixture was incubated at 37 °C for 5 min. Free D-alanine generated in this

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reaction was detected using the method described by Frere et al. (1976), and compared with a

191

standard D-alanine solution using a Multiskan Spectrum-1500 spectrophotometer (Thermo

192

Scientific, Nyon-1, Switzerland) set at 460 nm. (Frere et al., 1976). The kinetic parameters for

193

this assay were analyzed from the linear regression of the double reciprocal plot (Lineweaver and

194

Burk, 1934).

195

.

196

2.8. Kinetic study of the interaction of soluble enzymes with penicillin

197

The acylation rate k2/K (i.e. the rate of formation of acyl-enzyme complex with sub-

198

saturating concentration of the substrate) was obtained from time course study of enzyme-

199

substrate complex formation with Bocillin-FL (Chambers et al., 1994; Chowdhury et al., 2010).

200

The sPBP5 and smPBP5 were incubated with different concentration of Bocillin-FL (substrate)

201

for 30, 60, 90 and 120s. To stop the reaction, denaturing buffer was added and samples were

202

analyzed in 12% SDS-PAGE. The labeled proteins were quantified by Typhoon FLA 7000 (GE

203

Healthcare Bio-Sciences AB, Uppsala, Sweden).

204 205

The deacylation rate constant (k3) of soluble enzymes were determined by plotting the

206

value of remaining Bocillin-FL versus time (Di Guilmi et al., 2000; Chowdhury et al., 2010). In

207

brief, the enzymes were incubated with Bocillin-FL (50 µM) at 35º C for 30 min. Penicillin G (3

208

mM) was added at t=0 and fluorescent intensity was measured by removing the aliquots time to

209

time and subsequently denaturing the protein.

210 211

2.9. Assay of beta-lactamase activity of soluble enzymes

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The beta-lactam hydrolysis of sPBP5 and smPBP5 were determined with different groups

213

of beta-lactam antibiotics in the presence of 10 mM phosphate buffer (pH 7.4). The purified

214

proteins were incubated with the beta-lactam antibiotics at different concentrations and the

215

hydrolysis was monitored at respective wavelengths by using BioSpectrometer kinetic

216

(Eppendorf, Hamburg, Germany). The molar extinction coefficients for turnover of penicillin

217

and nitrocefin were ∆ε232 = -800 M-1cm-1 and ∆ε482 = 15,000 M-1cm-1 respectively (Urbach et al.,

218

2008).

219 220

2.10. In silico analysis of smPBP5

221

A constrained based homology program MODELLER 9v12 was adopted to build a 3D

222

model of the smPBP5 (Sali and Blundell, 1993). For model building, a PDB coordinate, 1NZO

223

chain A [The crystal structure of wild type PBP5 from E. coli, (Nicholas et al., 2003)] was used

224

as a template. The final model was validated through PROCHECK (Laskowski et al., 1993) and

225

Verify-3D (Luthy et al., 1992). To distinguish structural organization of the active-site

226

orientation, the active site of smPBP5 was superimposed to that of sPBP5 through Swiss PDB

227

deep view program and the result was analyzed in PyMol visualization tool. The active-site

228

groove volume of the enzyme was measured through CASTp calculation (Dundas et al., 2006).

229 230

2.11. Secondary structure determination of soluble proteins

231

To compare the overall folding of sPBP5 and smPBP5, Circular Dichroism (CD)

232

spectroscopy was done in the "far-UV" spectral region (200-240 nm) for determination of the

233

structural distribution of α -helix, β-sheet and random coil in each protein. Spectral data of the

234

samples were collected at different temperatures (i.e. 28, 35 and 40° C) with a 0.2 nm step

235

resolution, 1 s time constant and 10 milli degrees sensitivity at a 2.0 mm spectral bandwidth,

236

with a scanning speed of 50 nm min_1. Before, spectral data collection, all the soluble proteins

237

were diluted to same concentration (0.1 mg ml-1). Corrected spectra were obtained by subtracting

238

the solvent spectrum. To reduce noise and random instrumental error, an average spectrum was

239

compiled from four successive accumulations over a range of 200–240 nm. The percentage of α-

240

helix, β-sheet and random coil were calculated using program k2d3 software (Louis-Jeune et al.,

241

2012). This method compares the secondary structure of unknown protein with known data set

242

and analyzes the percentage of secondary structure of the sample protein.

243 244

Simultaneously, the secondary structure of sPBP5 (1NZO) and smPBP5 were also

245

verified by using two independent algorithms, PREDICT PROTEIN (Rost et al., 2004) and

246

PSIPRED (Jones, 1999).

247 248 249 250 251 252 253

254 255

3.0. Results

256

3.1. Site-directed mutagenesis at the ‘Ω type loop’ affects the cell shape maintaining ability of

257

PBP5

258

The strain CS703-1, derived from E. coli (CS109), lacks seven non-essential PBPs, and is

259

morphologically deformed (Nelson and Young, 2001; Ghosh et al., 2006). In trans expression of

260

PBP5 in this deletion mutant restores cell shape to near normal, i.e. rod shaped (Ghosh and

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Young 2003; Nelson and Young, 2000). To check whether the alteration in the ‘Ω type loop’ of

262

PBP5, i.e. the Leu182Glu mutation, could affect its ability to revert the abnormal cell shape of

263

CS703-1, the deformities of CS703-1 cells were checked upon ectopic expression of both PBP5

264

and PBP5L182E. Surprisingly, after complementing with PBP5 and its L182E mutant

265

individually in CS703-1, the levels of abnormalities in the cell shape were reduced to about 16%

266

and 75% respectively, as compared to the strain without any complementation (Fig. 1; Table.

267

S1). As the genes encoding PBP5 and PBP5L182E were cloned in pBAD18, the strain CS703-1

268

with pBAD18 was taken as a control.

269 270

To compare the cellular expression level and stability of PBP5L182E with that of wild

271

type PBP5, both PBP5 and PBP5L182E were expressed under the control of arabinose promoter

272

and isolated from the membrane of CS703-1 after 12 hrs of incubation at 37°C. It is noteworthy

273

to mention that the expression level and the stability of the proteins could have been checked by

274

immune-blotting technique. However, the similar procedure was not employed here because the

275

antibodies developed against PBP5 may also cross-react with DacD, which was present in the

276

CS703-1 cells, and it shares considerable identity with PBP5 (Potluri et al., 2010). Moreover, for

277

assessing the stability of the PBP5 and its mutant immune-blotting might not be the ideal choice,

278

because the PBP5 antibodies, in addition to binding to the active protein, can also bind to its

279

inactive variant. Therefore, it would be more meaningful to assess the in vivo expression for a

280

considerable interval of time and the stability of these proteins by labeling them subsequently

281

with a substrate like, Bocillin-FL. Upon labeling the equal amount of each protein with Bocillin-

282

FL (Fig. 2a), the similar intensities of the bound Bocillin-FL were found associated to PBP5 and

283

PBP5L182E indicating that both the PBPs were active to the same extent during their expression

284

in CS703-1. The overall results indicate that the mutation L182E in PBP5 almost aborted its

285

ability to restore the normal rod shape in E. coli CS703-1 strain.

286 287

3.2. The PBP5L182E can partially maintain the intrinsic beta-lactam resistance in E. coli

288

It is known that the deletion of PBP5 sensitizes E. coli cells to beta-lactam antibiotics.

289

Ectopic complementation of PBP5 restores the lost beta-lactam resistance of AM15-1 (PBP5

290

deletion mutant of CS109, an E. coli K-12 strain), since PBP5 possibly traps beta-lactams

291

(Sarkar et al., 2010). To check whether the Leu182Glu mutation can alter the intrinsic beta-

292

lactam resistance property of PBP5, the beta-lactam sensitivity of AM15-1 was tested by

293

expressing the PBP5L182E ectopically, using 0.0005% arabinose. It was observed that, unlike

294

the wild type, PBP5L182E was not able to restore the lost beta-lactam resistance of AM15-1

295

completely (Table. 2). The result signifies that PBP5L182E partially lost its ability to maintain

296

intrinsic beta-lactam resistance in E. coli. It can be speculated that PBP5 might have lost its

297

ability to exert efficient DD-CPase activity in vivo, which is required for maintaining cell shape.

298

It also retains its ability to bind beta-lactams, although the binding ability to beta-lactams may

299

have been reduced.

300 301

3.3. The smPBP5 exhibits poor DD-CPase activity

302

To know whether PBP5L182E had retained the ability to exert DD-carboxypeptidase

303

activity it was mandatory to analyze the biochemical nature of PBP5L182E in vitro and to

304

compare the same with the wild type PBP5. In order to verify the biochemical function of

305

PBP5L182E, the soluble form of it (smPBP5) was generated. Though PBP5 is a membrane

306

bound protein, for ease of purification, both the genes encoding sPBP5 and smPBP5 were cloned

307

in pET28a(+)and finally the proteins were purified by Ni-NTA affinity chromatography. The

308

concentrations of the proteins produced were ~1 mg ml-1. The molecular mass of the purified

309

proteins were ~44.0 kDa and both the enzymes were active as confirmed by labeling them with

310

Bocillin-FL and subsequent visualization in 12% SDS-PAGE (Fig. 2). The sPBP5 and smPBP5

311

were checked for its DD-CPase activity. Both the artificial peptidoglycan substrate (Nα,Nє-

312

diacetyl-Lys-D-Ala-D-Ala or, AcLAA) and a peptidoglycan mimetic pentapeptide substrate (L-

313

Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala or, AGLAA) were used (Chowdhury et al., 2012) as substrates

314

for the assay. The turnover numbers for AcLAA and AGLAA, i.e., kcat value of smPBP5

315

indicated ~10 fold lower product formation by smPBP5 as compared to sPBP5 (Table. 3a).

316

Therefore, the result indicates that indeed smPBP5 has lost the ability to exert in vitro DD-CPase

317

activity for both AcLAA and AGLAA substrates as compared to sPBP5, which is consistent with

318

the result obtained from in vivo study. Hence, we can infer that mutation of L182E not only

319

affects in vivo physiology of PBP5, but also the same is reflected in vitro.

320 321

3.4. Penicillin acylates smPBP5 at a rate that is little lesser as compared to sPBP5

322

To check the penicillin-binding efficacies of sPBP5 and the smPBP5, kinetic parameters

323

for acylation and deacylation were determined with fluorescent penicillin, Bocillin-FL by

324

calculating acylation and deacylation rate constants. The resultant rate of acylation (k2/K) of

325

smPBP5 for Bocillin was ~40% lower than that of sPBP5 (Table. 3b) indicating that the L153E

326

mutation affected the penicillin-binding efficiency of smPBP5. Further, the rate of dissociation

327

of penicillin from these enzymes, as determined by calculating the deacylation rate constant (k3),

328

showed ~10% lower rate in smPBP5 than sPBP5 (Table. 3b). Therefore, it can be stated that

329

L153E mutation has reduced the rate of penicillin-binding to sPBP5 by 40% without

330

significantly affecting its dissociation.

331 332

3.5. The L153E mutation doesn’t alter the beta-lactamase activity of sPBP5 in vitro

333

The glutamic acid at the Ω-loop of class A beta-lactamase is believed to provide water

334

molecule for the hydrolysis of beta-lactam antibiotics (Banerjee et al., 1998 and Sun et al.,

335

2003). To check whether the replacement of Glu at Leu153 in the purified soluble construct of

336

PBP5 could change in beta-lactamase activity, the hydrolysis of both penicillin and

337

cephalosporin group antibiotics were studied. However, there was no substantial difference in the

338

hydrolysis pattern of penicillin and nitrocefin by smPBP5 as compared sPBP5 (data not shown).

339 340

3.6. Replacement of Leu153 to Glu disrupt the association of the key residues in the sPBP5

341

active-site

342

To understand the reason behind the lower DD-CPase activity of smPBP5, both

343

biophysical and in silico studies were conducted. The structural model of the same was built to

344

compare with the crystal structure of wild type PBP5. The similarities in the secondary structure

345

analyses of the soluble proteins by both CD and in silico prediction (Fig. S1) revealed that there

346

was no significant change in overall folding of smPBP5 (Table. 4). In addition, both the proteins

347

(sPBP5 and smPBP5), were apparently stable within a physiological temperature range of 28 to

348

40 °C as revealed by the thermal stability assessment through CD (Fig. S2). However, the

349

superimposition study of the residues present in catalytic-sites exhibited a drastic deviation of the

350

γ-OH of both Ser44 and Ser110 and ε-NH2 of Lys47 residue (Fig. 3). In smPBP5, both γ-OH of

351

Ser44 and ε-NH2 of Lys47 are moved away from each other by 1.90 Ǻ and 2.18 Ǻ, respectively.

352

Thereby, the distance between Ser44 and Lys47 has been increased (Fig. 3) that prevents the H-

353

bond formation. In addition, the γ-OH of Ser110 has also deviated by 2.14 Ǻ creating an inter-

354

atomic distance of 4.2 Ǻ between Ser110 and Lys213 in smPBP5 whereas it is 2.7 Ǻ in case of

355

sPBP5 (Fig. 3). As a result, the higher distance between Ser110 and Lys213 might affect the H-

356

bond formation in smPBP5. This may interfere in the deacylation of peptidoglycan mimetic

357

substrates while exerting DD-CPase activity (Sauvage et al., 2008), though the deacylation of

358

Bocillin is not much affected (Table. 3b). Due to the adverse changes in the organization of the

359

key residues in smPBP5, the groove volume of the active-site has been decreased by almost 200

360

Ǻ3, which is the most likely reason for its lower ability to catalyse peptidoglycan mimetic

361

substrates (Fig. 4).

362 363 364 365 366 367

368 369 370 371

4.0. Discussion:

372

4.1. The possible reason for the functional limitation of L182E mutant of PBP5

373

The expression of PBP5 offers sufficient DD-CPase activity to preserve the consistency

374

in the cell morphology (Nelson and Young, 2001) and to maintain the intrinsic beta-lactam

375

resistance property in E. coli (Sarkar et al., 2011). The partial inability of PBP5L182E to reverse

376

the cell shape oddities of CS703-1 to normal suggests impairment in its DD-CPase activity (Fig.

377

1). To verify the physiological reason of the particular mutation, biochemical activity of smPBP5

378

has been examined, where it has shown a ~10 fold lower DD-CPase activity with the

379

peptidoglycan mimetic pentapeptide substrate than sPBP5 (Table. 3a). Therefore, it seems

380

obvious that L182E mutation of PBP5 drastically affect the DD-CPase activity, both in vivo and

381

in vitro.

382 383

Additionally, we find that PBP5 with L182E mutation lacks the ability to reverse the lost

384

intrinsic beta-lactam resistance in ∆PBP5 mutant of E. coli (Table. 2). It has been speculated

385

earlier that the high copy number PBP5 traps beta-lactam antibiotics to shield over other

386

essential PBPs (Sarkar et al., 2010). So, if the rate of acylation of PBP5 were lowered towards

387

beta-lactam substrates, the ability of PBP5 to act as traps would supposedly diminish. Indeed,

388

the rate of acylation for smPBP is ~40% lower for the penicillin substrate than sPBP5 (Table.

389

3b). Therefore, such reduction in the acylation rate might be the contributor for the partial

390

inability of PBP5L182E to reverse intrinsic beta-lactam resistance.

391 392

Definitely, the speculative insight into the mechanism behind the inefficiency of smPBP5

393

to exert low DD-CPase activity, especially towards peptidoglycan mimetic substrate, lies into the

394

packing of the active site. The distance between γ-OH of Ser44 and ε-NH2 of Lys47 is increased

395

in smPBP5, which may result in an unsuccessful interaction and possibly weakens the

396

nucleophilicity of the active-site Ser44 (Davies et al., 2001 and Stefanova et al., 2002). Thus,

397

greater distance may also hinder the acylation rate of the peptidoglycan mimetic substrates

398

(Table. 3a). Moreover, Ser110, which helps in hydrolyzing peptide substrate (s) by polarizing

399

water molecules with the help of Lys213 (Nicola et al., 2005), also undergoes deviation in

400

smPBP5 and thereby raising the distance between γ-OH of Ser110 and ε-NH2 of Lys213. Such

401

alteration in distance might hamper the stability of the acyl-enzyme complex during deacylation

402

(Malhotra and Nicholas, 1992). Furthermore, as there is a reduction in the active-site grove

403

volume by ~25% in the smPBP5 than in sPBP5, the space at the binding pocket is possibly

404

inadequate for proper fitting of the pentapeptide substrate (Fig. 4). Hence, the release of terminal

405

D-alanine may be less during DD-CPase reaction, lowering the enzymatic efficiency of smPBP5.

406

However, the interaction of the smPBP5 with the relatively smaller substrate Bocillin-FL is

407

barely affected as it can fit comfortably into the groove (Table. 3b). A similar effect has been

408

reported for E. coli DacD (Chowdhury et al., 2012). Therefore, the present observation is in line

409

with our earlier speculation that though there is no remarkable difference in overall folding of the

410

enzyme, the activity of the PBP5 towards the substrate depends not only on the orientation of the

411

active-site residues but also on the active-site groove-volume. In a nutshell, it can be stated that

412

the discrete organization of the active-site residues in smPBP5 may be attributed to two big

413

deflections, one between Ser44 and Lys47 and another between Ser110 and Lys213, which

414

adversely affect their orientation and the active-site groove volume. This in turn impedes the

415

enzymatic activity both in vivo and in vitro. Therefore, we conclude that Leu182 plays a pivotal

416

role in maintaining the physiology of E. coli PBP5.

417 418 419 420 421 422

Acknowledgement

423

This work was supported in part by two different grants. One from the Department of

424

Biotechnology (DBT-NE program), Government of India and another from Sponsored Research

425

and Industrial Consultancy (SRIC) SGIRG seed Grant to ASG. MD and DK were supported by

426

Institute Fellowship of IIT Kharagpur and AB was Council of Scientific and Industrial Research,

427

Govt. of India.

428 429 430 431 432 433 434 435 436

437 438 439 440

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441

Baquero, M.R., Bouzon, M., Quintela, J.C., Ayala, J.A. & Moreno, F. (1996). dacD, an

442

Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with DD-

443

carboxypeptidase activity. J Bacteriol 178, 7106-7111

444

Banerjee, S., Pieper, U., Kapadia, G., Pannell, L.K. & Herzberg, O. (1998). Role of the Ω-

445

loop in the activity, substrate specificity, and structure of class A β-lactamase. Biochemistry

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37, 3286–3296.

447

Chakraborty, S., (2012). An Automated Flow for Directed Evolution Based on Detection of

448

Promiscuous Scaffolds Using Spatial and Electrostatic Properties of Catalytic Residues.

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Chambers, H.F., Sachdeva, M.J. & Hackbarth, C.J. (1994). Kinetics of penicillin binding to penicillin-binding proteins of Staphylococcus aureus. Biochem J 301, 139–144.

452

Chowdhury, C., Nayak, T.R., Young, K.D. & Ghosh, A.S. (2010). A weak DD-

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carboxypeptidase activity explains the inability of PBP 6 to substitute for PBP 5 in

454

maintaining normal cell shape in Escherichia coli. FEMS Microbiol Lett 303, 76–83.

455

Chowdhury, C., Kar, D., Dutta, M., Kmar, A. & Ghosh, A.S. (2012). Moderate deacylation

456

efficiency of DacD explains its ability to partially restore beta-lactam resistance in

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Escherichia coli PBP5 mutant. FEMS Microbiol Lett 337, 73–80.

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Davies, C., Whites, S.W. & Nicholas, R.A. (2001). Crystal Structure of a Deacylation-defective Mutant of Penicillin-binding Protein 5 at 2.3-Å Resolution. J BiolChem 276, 616–623.

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Di Guilmi, A.M., Mouz, N., Petillot, Y., Forest, E., Dideberg, O. & Vernet, T. (2000).

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462

mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry.

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Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y. & Liang, J. (2006). CASTp:

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466

of functionally annotated residues. Nucleic Acids Res 34, W116–8.

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Frere, J.M., Leyh-Bouille, M., Ghuysen, J.M., Nieto, M. & Perkins, H.R. (1976). Exocellular

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DD-carboxypeptidases-transpeptidases from Streptomyces. Methods Enzymol 45, 610–636.

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Ghosh, A.S., Chowdhury, C. & Nelson, D.E. (2008). Physiological functions of D-alanine

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carboxypeptidases in Escherichia coli. Trends Microbiol 16, 309–317.

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Ghosh, A.S., Melquist, A.L. & Young, K.D. (2006). Loss of O-antigen increases cell shape

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abnormalities in penicillin-binding protein mutants of Escherichia coli. FEMS Microbiol Lett

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Ghosh, A.S. & Young, K.D. (2003). Sequences near the Active Site in Chimeric Penicillin

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Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli. J Bacteriol 185,

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2178–2186.

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Ghuysen, J.M. (1991). Serine β-lactamases and penicillin-binding proteins. Annu Rev Microbiol 45, 37–67.

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González-Leiza, S.M., de Pedro, M.A. & Ayala, J.A. (2011). AmpH, a bifunctional DD-

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endopeptidase and DD-carboxypeptidase of Escherichia coli. J Bacteriol 193, 6887-6894.

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Holtje, J.V. (1998). Growth of the stress-bearing and shape-maintaining murein sacculus of

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Escherichia coli. Microbiol Mol Biol Rev 62, 181–203.

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Jones, D.T. (1999). Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292, 195–202.

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Korat, B., Mottl, H. & Keck, W. (1991). Penicillin-binding protein 4 of Escherichia coli:

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molecular cloning of the dacB gene, controlled overexpression, and alterations in murein

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Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. (1993). PROCHECK: a

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Lineweaver, H. & Burk, D. (1934). The determination of enzyme dissociation constants. J Am Chem Soc 56, 658–666.

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Louis-Jeune, C., Andrade-Navarro, M.A. & Perez-Iratxeta, C. (2012). Prediction of protein

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Luthy, R., Bowie, J.U. & Eisenberg, D. (1992). Assessment of protein models with threedimensional profiles. Nature. 356, 83–85.

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Malhotra, K.T. & Nicholas, R.A. (1992). Substitution of lysine 213 with arginine in penicillin-

500

binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without

501

affecting penicillin binding. J Biol Chem 267, 11386–91.

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Nelson, D.E. & Young, K.D. (2000). Penicillin Binding Protein 5 Affects Cell Diameter, Contour and Morphology of Escherichia coli. J Bacteriol 183, 1714–1721.

504

Nelson, D.E. & Young, K.D. (2001). Contributions of PBP 5 and DD-Carboxypeptidase

505

Penicillin Binding Proteins to Maintenance of Cell Shape in Escherichia coli. J Bacteriol

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183, 3055–3064.

507

Nelson, D.E., Ghosh. A.S., Paulson, A.L. & Young, K.D. (2002). Contribution of membrane-

508

binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform

509

cellular morphology of Escherichia coli. J Bacteriol 184, 3630–3639.

510

Nicola, G., Fedarovich, A., Nicholas, R.A. & Davies, C. (2005). A large displacement of the

511

SXN motif of Cys115-modified penicillin-binding protein 5 from Escherichia coli. Biochem

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J 392, 55–63.

513

Nicola, G., Peddi, S., Stefanova, M., Nicholas, R.A., Gutheil, W.G. & Davies, C. (2005).

514

Crystal Structure of Escherichia coli Penicillin-Binding Protein 5 Bound to a Tripeptide

515

Boronic Acid Inhibitor: A Role for Ser-110 in Deacylation. Biochemistry 44, 8207–8217.

516

Nicholas, R.A., Krings, S., Tomberg, J., Nicola, G. & Davies, C. (2003). Crystal structure of

517

wild-type penicillin-binding protein 5 from Escherichia coli: implications for deacylation of

518

the acyl-enzyme complex. J Biol Chem 278, 52826–33.

519

Nilsen T, Ghosh A.S., Goldberg M. B.

& Young K.D. (2004). Branching sites and

520

morphological abnormalities behave as ectopic poles in shape-defective Escherichia coli.

521

Mol Microbiol 52 (4), 1045–1054

522

Potluri, L., Karczmarek , A., Verheul, J., Piette, A., Wilkin, J.M., Werth, N., Banzhaf, M.,

523

Vollmer, W., Young, K.D., Nguyen-Distèche, M. & den Blaauwen, T. (2010). Septal and

524

lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli,

525

requires substrate recognition and membrane attachment. Mol Microbiol 77, 300-323.

526 527 528 529

Rost, B., Yachdav, G. & Liu, J. (2004). The PredictProtein server. Nucleic Acids Res 32, W321–W326. Sali, A. & Blundell, T.L. (1993). Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234, 779–815.

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Sarkar, S.K., Chowdhury, C. & Ghosh, A.S. (2010). Deletion of penicillin-binding protein 5

531

(PBP5) sensitises Escherichia coli cells to beta-lactam agents. Int J Antimicrob Agents 35,

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244–249.

533

Sarkar, S.K., Dutta, M., Chowdhury, C., Kumar, A. & Ghosh, A.S. (2011). PBP5, PBP6 and

534

DacD play different roles in intrinsic β-lactam resistance of Escherichia coli. Microbiology

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Sauvage, E., Powell, A.J., Heilemann, J., Josephine, H.R., Charlier, P., Davies, C. & Pratt,

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R.F. (2008). Crystal Structures of Complexes of Bacterial DD-Peptidases with

538

Peptidoglycan-Mimetic Ligands: The Substrate Specificity Puzzle. J Mol Biol 381, 383–393.

539

Stefanova, M.E., Davies, C., Nicholas. R.A. & Gutheil, W.G. (2002). pH, inhibitor, and

540

substrate specificity studies on Escherichia coli penicillin-binding protein 5. Biochim.

541

Biophys.Acta. 1597, 292–300.

542

Sun, T., Nukaga, M., Mayama, K., Braswell, E.H. & Knox, J.R. (2003). Comparison of β-

543

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544

oxacillinase. Protein Sci 12, 82–91.

545

Urbach, C., Fastrez, J. & Soumillion, P. (2008). A new family of cyanobacterial penicillin-

546

binding proteins: a missing link in the evolution of class A betalactamases. J Biol Chem 283,

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548

van der Linden, M.P., de Haan, L., Dideberg, O. & Keck, W. (1994). Site-directed

549

mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia

550

coli. Biochem J 303, 357–362.

551

Zhang, W., Shi, Q., Meroueh, S.O., Vakulenko, S.B. & Mobashery, S. (2007). Catalytic

552

mechanism of penicillin-binding protein 5 of Escherichia coli. Biochemistry 46, 10113–21.

553

Zhao, G., Meier, T.I., Kahl, S.D., Gee, K.R. & Blaszczak, L.C. (1999). BOCILLIN FL, a

554

sensitive and commercially available reagent for detection of penicillin-binding proteins.

555

Antimicrob Agents Chemother 43, 1124–1128.

556 557 558 559 560 561 562 563 564 565 566 567 568 569 570

571 572

Figure Legends

573

Fig. 1. Cell shape of CS703-1 with the expression of PBP5L182E. Row 1: without aztreonam

574

and Row 2: with aztreonam (5 µg ml-1). From Left to right Column 1: CS703-1 with pBAD18

575

cam; Column 2: CS703-1 with pPJ5 and Column 3: CS703-1 with pPJ5L182E

576 577

Fig. 2a. SDS-PAGE analysis of membrane proteins: Lane 1, 3 and 4 show the proteins that are

578

stained with comassie blue stain; Lane 5, 7 and 8 are the Bocillin-binding of the membranes of

579

CS703-1/pBAD, CS703-1/pPJ5 and CS703-1/pPJ5L182E, respectively. Lanes 2 and 6 are for

580

Protein molecular mass marker.

581

2b. SDS-PAGE (12%) analysis of the purified soluble enzymes: Lane 1: Protein molecular mass

582

marker, Lane 2: purified sPBP5; Lane 3: purified smPBP5; Lane 4 and Lane 5 are purified

583

sPBP5 and smPBP5 labeled with Bocillin-FL, respectively.

584 585

Fig. 3. Superimposition of the active-site residues of sPBP5 (1NZO) over smPBP5 (RMSD value

586

is 0.91 Å). The residues of sPBP5 and smPBP5 are colored in ‘Green’ and ‘Magenta’,

587

respectively. In smPBP5, the γ-OH of Ser110, γ-OH of Ser44 and ε-NH2 of Lys47 are twisted

588

from their original position by 100.28°, 85.34° and 125.80°, respectively.

589 590

Fig. 4. The surface-view of (a) 1NZO and (b) modeled smPBP5. The active-site groove volumes

591

of 1NZO and smPBP5 are shown in yellow color. (a) 1NZO = 960 Å3 and smPBP5= 782 Å3

592

593 594 595 596 597 598

Table 1. List of E. coli strains and plasmids

599

600 601 602 603 604 605 606 607

E. coli strains

Genotypes

Source or References

CS109

W1485 rpoS rph

C. Schnaitman

AM15-1

CS109∆PBP5

Sarkar et al., 2010

CS703-1

CS109∆PBP1, 4, 5, 6, 7, AmpC and AmpH

Nelson et al., 2002

Plasmids

Features

Source or References

pPJ5

E. coli PBP5 cloned in pBAD18 Cam

Kevin D. Young

pT7-cPBP5

E. coli sPBP5 cloned in pT7-7k

Robert A. Nicholas

pET5

E. coli sPBP5 cloned in pET28a(+)

This study

pPJ5L182E

E. coli PBP5L182E cloned in pBAD 18 Cam

This study

pTS5LE

E. coli sPBP5L153E (smPBP5) cloned in pT7-7k

This study

pET5LE

E. coli sPBP5L153E (smPBP5) cloned in pET28a(+)

This study

608 609 610 611 612 613

Table 2. Beta-lactam sensitivity of E. coli strains carrying pPJ5L182E

614

E. coli strains CS1O9 AM15-1 AM15-1/pPJ5 AM15-1/pPJ5L182E 615 616 617 618 619 620 621 622 623 624 625 626

PenicillinG 250 64 250 125

MIC (µg ml-1) Piperacillin Cefadroxil 2 32 0.5 8 2 32 1 16

Cefaclor 4 0.5 4 2

627 628 629 630 631

Table 3. Kinetic parameters of the enzymes with pentapeptides (a) and penicillin (b)

632

(a) sPBP5

smPBP5

Km (mM)

kcat (s-1)

Km (mM)

kcat (s-1)

L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala

0.434±0.06

1.97±0.2

0.042±0.002

0.194±0.06

Nα,Nє-diacetyl-Lys-D-Ala-D-Ala

0.538±0.03

2.45±0.4

0.035±0.007

0.2±0.04

Substrate

633 634

(b) sPBP5

smPBP5

Substrate

Penicillin (Bocillin FL) 635 636 637 638 639 640 641 642

k2/K (M-1s-1)

k3 (s-1)

k2/K (M-1s-1)

k3 (s-1)

730±17.9

14.9±1.7

463±13.3

16.4±6.5

643 644 645 646 647 648 649 650

Table 4. Percentage distribution of secondary structures obtained by using different

651

techniques Different

Circular Dichroism

Techniques Secondary

α-Helix

β-Sheet

Random coil

α-Helix

β-Sheet

Random coil

sPBP5

22.60%

28.71%

48.69%

24.73%

29.67%

45.60%

smPBP5

21.41%

27.39%

51.2%

24.45%

29.95%

45.60%

structures

652 653 654 655

PredictProtein server

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A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance.

Penicillin-binding protein 5 (PBP5), a dd-carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance in E. coli. A strain lacking PBP...
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