Cloning of feather-degrading minor extracellular protease from Bacillus cereus DCUW: dissection of the structural domains Abhrajyoti Ghosh, 1,2 3 Krishanu Chakrabarti 1 and Dhrubajyoti Chattopadhyay 1,2 Correspondence Dhrubajyoti Chattopadhyay djcbcg@caluniv.ac.in 1 Department of Biochemistry, University of Calcutta, India 2 Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, India Received 15 January 2009 Revised 22 February 2009 Accepted 4 March 2009 Bacterial extracellular proteases play an important role in cell survival and cell–cell communication. A high-molecular-mass minor extracellular protease (Vpr) from a feather- degrading bacterium, Bacillus cereus DCUW, has been reported by our laboratory. In the present study, we cloned and expressed Vpr in Escherichia coli. Complete nucleotide sequencing of this gene predicted that the protease is a member of the serine protease family, and SMART domain analysis revealed that the protease consists of an N-terminal signal sequence for secretion, a subtilisin_N sequence that is a signature for N-terminal processing, a catalytic S_8 peptidase domain, and finally a long C-terminal protease-associated (PA) region containing nine intrinsically disordered subdomains. Four truncated constructs of the Vpr protease were cloned and expressed in E. coli. We found that the catalytic domain (amino acid residues 172–583) is sufficient for protease activity. Maturation of the Vpr protease needed both N-terminal and C- terminal processing. We have demonstrated that the oligomerization property is associated with the C-terminal protease-associated domain and also shown that the substrate-binding specificity to raw feather resides in this domain. INTRODUCTION Secretory proteins in bacteria are known to execute several very important ‘remote-control’ functions, such as nutrient cycling and utilization, cell-to-cell communication and detoxification of the extracellular environment, and they may also behave as weapons against potential competitors. Most of these secretory proteins are synthesized as a preproprotein with an N-terminal signal peptide, which is required to target these proteins to the preprotein translo- case in the membrane and initiate the translocation process (Pugsley, 1993). Membrane-bound type I signal peptidases (Palacin et al., 2002) remove this signal peptide to release the mature secretory protein from the trans side of the membrane either during or shortly after translocation (Dalbey et al., 1997). Statistical studies of sequences surrounding the signal peptidase cleavage site led to the formulation of the 21, 23 or Ala-X-Ala rule, defining the preferred residues (i.e. Ala) at the 21 and 23 positions relative to the cleavage site as critical determinants for signal peptide recognition and cleavage (von Heijne, 1983, 1985). Many bacterial proteolytic enzymes are synthesized as inactive precursors, or zymogens, to prevent unwanted protein degradation and to enable spatial and temporal regulation of proteolytic activity (Khan & James, 1998). Biochemical studies of the activation mechanism of individual proteases have provided insights into their physiological functions. In the Bacillus system, every extracellular protease is found to be synthesized as a preproenzyme in the cytoplasm and is processed to a mature enzyme in the extracellular milieu. The activa- tion mechanism of subtilisin from Bacillus subtilis has been well studied. The zymogen form of subtilisin is converted to an active form via intramolecular auto- processing (Ohta & Inouye, 1990) in the extracellular milieu (Inouye, 1991). The prepeptide has been shown to guide the proper folding of subtilisin in vivo and in vitro (Strausberg et al., 1993). Most extracellular proteases in the Bacillus system seem to be activated by auto-processing. The limited proteolysis actually dictates the auto-processing among proteases in the extracellular medium. Moreover, studies of the activa- tion of different secreted enzymes from their pre-enzyme forms have shown that proteases have a definite role in Abbreviation: DLS, dynamic light scattering. 3Present address: Max Plank Institute for Terrestrial Microbiology, Karl- von-Frisch-Strasse, D-35043 Marburg, Germany. The GenBank/EMBL/DDBJ accession number for the sequence of the vpr gene is EU626488. Microbiology (2009), 155, 2049–2057 DOI 10.1099/mic.0.027573-0 027573 G 2009 SGM Printed in Great Britain 2049