International Journal of Biological Macromolecules 58 (2013) 301–309 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo ur nal homep age: www.elsevier.com/locate/ijbiomac Structural and functional studies on urease from pigeon pea (Cajanus cajan) Anuradha Balasubramanian a , Vishnuprabu Durairajpandian b , Sagadevan Elumalai c , Narayanasamy Mathivanan c , Arasambattu Kannan Munirajan b , Karthe Ponnuraj a,∗ a Centre of Advanced Study in Crystallography and Biophysics University of Madras, Guindy Campus, Chennai 600025, India b Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences University of Madras, Taramani Campus, Chennai 600113, India c Centre of Advanced Study in Botany University of Madras, Guindy Campus, Chennai 600025, India a r t i c l e i n f o Article history: Received 28 January 2013 Received in revised form 30 March 2013 Accepted 10 April 2013 Available online 24 April 2013 Keywords: Urease Pigeon pea Sequence Crystal structure Antifungal Insecticidal a b s t r a c t Urease is an enzyme that catalyzes the hydrolysis of urea, forming ammonia and carbon dioxide, and is found in plants, microorganisms and invertebrates. Although plant and bacterial ureases are closely related at amino acid and at the structural level, the insecticidal activity is seen only in the plant ureases. In contrast, both plant and bacterial ureases exhibit antifungal activity. These two biological properties are independent of its ureolytic activity. However, till date the mechanism(s) behind the insecticidal and fungicidal activity of ureases are not clearly understood. Here we report the crystal structure of pigeon pea urease (PPU, Cajanus cajan) which is the second structure from the plant source. We have deduced the amino acid sequence of PPU and also report here studies on its stability, insecticidal and antifungal activity. PPU exhibits cellulase activity. Based on the structural analysis of PPU and docking studies with cellopentoase we propose a possible mechanism of antifungal activity of urease. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Ureases (urea amidohydrolase EC 3.3.1.5) are nickel dependent enzymes, [1] widespread in plants, fungi and bacteria [2,3] that cat- alyze the hydrolysis of urea to form ammonia and carbon dioxide at a rate 8 × 10 17 times faster than the uncatalyzed reaction [4]. Plant and fungal ureases are homo-hexamers of 90 kDa subunits. In contrast, bacterial ureases are multimers of two or three sub- unit complexes. The plant, fungal and bacterial ureases show high sequence similarity and have highly conserved secondary struc- tures and similar catalytic mechanisms [3,5]. Although the amino acid sequences of plant and bacterial ureases are closely related, the insecticidal activity is seen only in the plant ureases [6]. Urease from Jack beans (JBU) is historically a very important protein. It was the first enzyme to be crystallized by James B. Sumner in 1926. Also it was the first enzyme found to contain metal nickel in its active site [1]. The insecticidal activity of plant urease was observed only in insects whose digestive system is based on cathepsin-like enzymes. The insecticidal activity was first observed in canatoxin, an isoform of JBU, [7] and later it was detected in jackbean and soybean seed specific urease [8]. The insects that depend on cathepsin B and D for ∗ Corresponding author. Tel.: +91 44 2220 2775; fax: +91 44 2230 0122. E-mail addresses: pkarthe@hotmail.com, karthe@unom.ac.in (K. Ponnuraj). their digestion like, the cowpea weevil Callosobruchus maculatus; the green stinkbug Nezara viridula; cotton stainer bug Dysdercus peruvianus and the kissing bug Rhodnius prolixus are vulnerable to urease at a concentration of 0.02–0.1% (w/w) [8]. Canatoxin, jack- bean and soybean ureases are hydrolyzed by cathepsin B and D enzymes to release an internal 10 kDa entomotoxic peptide termed pepcanatox [9]. A recombinant peptide similar to pepcanatox, known as jaburetox-2Ec also exhibited potent insecticidal prop- erties [10]. However, plant ureases failed to have its insecticidal effects on the insects relying on trypsin for their digestion, which is because the pepcanatox region is not intact [8,10] and this region is further cleaved into smaller fragments by trypsin. Till date, the mechanism of insecticidal activity of urease is not characterized completely but a lot of biochemical studies and crystal structure analysis of JBU have shown that the amphipathic  hairpin (Lys 290 - Glu 303 ) located within the 10 kDa region is likely responsible for this activity [10–12]. Besides insecticidal activity, plant ureases possess an antifun- gal activity and this property is observed in bacterial ureases also [13]. Canatoxin was previously reported to have antifungal activity against phytopathogenic fungi Macrophamina phaseolina, Sclerotium rofstii and Colletotrichum gloesporioides [14]. Jackbean and soybean ureases are also shown to have potent antifungal activity against Curvularia lunata, Fusarium solani, Penicillium her- guei, Trichoderma viride, Trichoderma pseudokoningii, Colletotrichum musae and Fusarium oxysporum [13]. Recently, cotton urease was 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.04.055