Association of a Zn2+ containing metallo β-lactamase with the anticancer antibiotic mithramycinJournal of Inorganic Biochemistry


Shibojyoti Lahiri, Amrita Panja, Dipak Dasgupta
Inorganic Chemistry / Biochemistry


Redefining Residential: Integrating Evidence-Based Practices

American Association of Children's

Emergence of New Delhi metallo-β-lactamase in Jerusalem, Israel

Tamar Lachish, Meital Elimelech, Natalie Arieli, Amos Adler, Jean-Marc Rolain, Marc Victor Assous

Max born medal and prize

The Institute of Physics

Type I β-lactamases ofEnterobacter cloacae and resistance to β-lactam antibiotics

M. T. Tejedor-Junco, M. González-Martín, P. Lupiola, Z. González-Lama


Association of a Zn2+ containing metallo β-l 064, e re dies han bin oth

E[M curs ligh mic iary structural levels. The enzyme activity decreases as a consequence to this rally oc inhibi with t e Mg2+ neopla cinoma 2+ types of β-lactamases, sisting exclusively of o-factors [22]. Broad es and resistance to a

Journal of Inorganic Biochemistry 142 (2015) 75–83

Contents lists available at ScienceDirect

Journal of Inorgan j ourna l homepage: www.e lsewide range of β-lactam antibiotics have made the design of clinically appropriate inhibitors for this class of enzymes very difficult.

Here, we have shown by means of spectroscopy and calorimetry

Maximilians-Universität, Schillerstr. 42, Munich, Germany. Tel.: +49 89 2180 75 775; fax: +49 89 2180 75 425. ⁎⁎ Correspondence to: D. Dasgupta, Biophysics and Structural Genomics Division, Saha[13]. Furthermore, MTR derivatives produced by combinatorial biosynthesis show lower toxicity and better pharmacological properties, used β-lactam antibiotics [21]. Among different class B β-lactamases make a unique group con

E[MBL]s which utilize one or two zinc ions as c substrate profiles, varying metal stoichiometri ⁎ Correspondence to: S. Lahiri, Zentrallabor für Proteinanalytik (ZfP), Ludwig-host of disorders besides cancer is observed as new uses and activities are ascribed to this generic drug [6–12]. High-throughput screening studies show that MTR, in spite of its small therapeutic window, is worth considering as potential lead compound for clinical application metalloenzymes. Zn containing metallo β-lactamases (E[MBL]) could, therefore, be a potential target of the drug. It is well established that a class of penicillin and penicillin derivative(s) resistant bacterial strains employ these enzymes to inhibit the bactericidal ability of commonlyInstitute of Nuclear Physics, 1/AF Sector-1, Bidhann

Tel.: +91 33 23370379x3506; fax: +91 33 23374637.

E-mail addresses: Shibojyoti.Lahiri@med.uni-muenche (D. Dasgupta). 1 Present address: Tata Consultancy Services, Plot N

Technology Park, Siruseri, Navalur 603103, India. 0162-0134/© 2014 Elsevier Inc. All rights reserved., and Paget's disease of ical use because of its application of MTR in a have been studying the bivalent metal ion (Zn ) binding ability of the antibiotic to inhibit metalloenzymes [18]. The broad objective is to evaluate the potential of MTR for the treatment of diseases involvingbones [1]. Presently, MTR has limited clin systemic toxicity. Nonetheless, resurgence inIntroduction

Mithramycin (MTR, Fig. 1) is a natu ed from Streptomyces plicatus [1], which transcription via reversible interaction the presence of bivalent metal ions lik sively used in the treatment of various myelogenous leukemia, testicular carthe enzyme. Our results suggest that the naturally occurring antibiotic MTR, a generic drug, has the potential as an E[MBL] inhibitor. © 2014 Elsevier Inc. All rights reserved. curring antibiotic obtaints both in vivo and in vitro he DNA minor groove, in [1–5]. MTR was extenstic diseases like chronic thereby enhancing the potential of this family of compounds as therapeutic agents [14–17].

Earlier, we have demonstrated the molecular basis of the mechanism of action of this antibiotic, where it is observed that the anionic antibiotic (pKa[MTR] = 5.0) alone binds to bivalent metal ions and it is the [antibiotic:metal ion] complexes that actually act as DNAbinding ligands at and above neutral pH [18–20]. In our laboratory we 2+Mithramycin conformational disruption arising from the formation of a ternary complex involving MTR, catalytic Zn2+ andMetalloenzyme

Metal ion binding strong association with subat both secondary and tertanticancer antibiotic mithramycin

Shibojyoti Lahiri ⁎, Amrita Panja 1, Dipak Dasgupta ⁎⁎

Biophysics Division, Saha Institute of Nuclear Physics, 1/AF Sector-1, Bidhannagar, Kolkata 700 a b s t r a c ta r t i c l e i n f o

Article history:

Received 14 June 2014

Received in revised form 3 October 2014

Accepted 3 October 2014

Available online 14 October 2014


Enzyme inhibition

Metallo β-lactamase

Pathogenic bacteria that ar the antibiotics. Current stu involved in the defense mec

B1 E[MBL] from Bacillus cereus (CD and fluorescence) and is with the Zn2+ ion containing this high affinity complex oc

CD spectroscopy, dynamicagar, Kolkata 700064, India. (S. Lahiri), o. 1/G1, SIPCOT Informationactamase with the

India sistant to β-lactam antibiotics mostly utilize serine β-lactamases to degrade have shown that different subclasses of metallo β-lactamases (E[MBL]) are ism of drug resistant bacteria. Here we report that the Zn2+ containing subclass ds to a naturally occurring anti-cancer drug mithramycin (MTR). Spectroscopic ermal titration calorimetry studies show that MTR forms a high affinity complex

BL]. Abolished interaction of MTR with apo E[MBL] suggests that the formation of due to the potential of MTR to bind bivalent metal ions like Zn2+. Furthermore, t scattering and differential scanning calorimetry studies indicate that the romolar dissociation constant leads to an alteration in the enzyme conformation ic Biochemistry v ie r .com/ locate / j inorgb iobased studies that MTR binds to E[MBL] from Bacillus cereus. The association leads to MTR-induced structural alterations in the enzyme that culminates in the inhibition of its activity via a reversible mixed inhibition type of mechanism. Finally, we have proposed a binding model of MTR at the Zn2+ containing active site of the protein. The and used without further purification. K2HPO4, KH2PO4, Trizma base, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Na-salt, availability of the drug stocks. MTR from each supplier and each batchwas spectroscopically checked for quality (from the ratio of abThe protein was extensively dialyzed against the experimental buffers, prior to each experiment and concentration was measured re o 76 S. Lahiri et al. / Journal of Inorganic Biochemistry 142 (2015) 75–83spectrophotometrically at 280 nm using molar absorption coefficient, ε= 30,500 M−1 cm−1 [24]. The purity of the commercial sample was checked in a 10% SDS-PAGE (Fig. S1). Apo E[MBL] was prepared as described earlier [25]. Apo E[MBL] was checked for its Zn2+ content using PAR under denaturing conditions as reported earlier [26]. Thesorbance values at 400 nm and 440 nm) prior to each experiment.