RESEARCH LETTER A weak DD-carboxypeptidase activity explains the inability of PBP 6 to substitute for PBP 5 in maintaining normal cell shape in Escherichia coli Chiranjit Chowdhury 1 , Tapas R. Nayak 1 , Kevin D. Young 2 & Anindya S. Ghosh 1 1 Department of Biotechnology, Indian Institute of Technology, Kharagpur, WestBengal, India; and 2 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Correspondence: Anindya S. Ghosh, Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India. Tel.: 1 91 3222 283798; fax: 1 91 3222 278707; e-mail: anindyain@yahoo.com Received 5 October 2009; revised 3 November 2009; accepted 15 November 2009. Final version published online 15 December 2009. DOI:10.1111/j.1574-6968.2009.01863.x Editor: Anthony George Keywords penicillin-binding proteins; morphology maintenance; DD-carboxypeptidase; Escherichia coli. Abstract Penicillin-binding protein (PBP) 5 plays a critical role in maintaining normal cellular morphology in mutants of Escherichia coli lacking multiple PBPs. The most closely related homologue, PBP 6, is 65% identical to PBP 5, but is unable to substitute for PBP 5 in returning these mutants to their wild-type shape. The relevant differences between PBPs 5 and 6 are localized in a 20-amino acid stretch of domain I in these proteins, which includes the canonical KTG motif at the active site. We determined how these differences affected the enzymatic properties of PBPs 5 and 6 toward b-lactam binding and the binding and hydrolysis of two peptide substrates. We also investigated the enzymatic properties of recombinant fusion proteins in which active site segments were swapped between PBPs 5 and 6. The results suggest that the in vivo physiological role of PBP 5 is distinguished from PBP 6 by the higher degree of DD-carboxypeptidase activity of the former. Introduction Of the 12 known penicillin-binding proteins (PBPs) in Escherichia coli, four are DD-carboxypeptidases (DD-CPases): PBPs 4, 5 and 6, and DacD (Holtje, 1998; Ghosh et al., 2008). These enzymes remove the terminal D-alanine from the pentapeptide side chains of muramic acid in peptidogly- can, and the resulting tetrapeptides cannot act as donors during the formation of peptide cross-links in the cell wall (Ghuysen, 1991). Although the DD-CPases are usually the most abundant PBPs in the cell, they are not essential for bacterial survival (Denome et al., 1999) and the in vivo purposes of these seemingly nonessential and redundant enzymes are mostly unknown. The exception to the above statement is the E. coli protein PBP 5, which helps maintain the normal morphology of this organism even in the absence of seven other PBPs (Nelson & Young, 2001). In the absence of PBP 5 by itself, the cells exhibit small morphological aberrations, but as more PBPs are deleted, the cells become considerably misshapen (Nel- son & Young, 2000, 2001). PBP 5 consists of two major domains, I and II, oriented almost at right angles to one another (Davies et al., 2001; Nicholas et al., 2003). The DD- CPase active site is located in domain I and is responsible for maintaining normal cell shape (Nelson et al., 2002; Ghosh & Young, 2003). Domain II is composed mostly of b-sheets and may lift the enzymatic domain away from the inner membrane and into the periplasm toward the peptidoglycan substrate (Nelson et al., 2002; Ghosh & Young, 2003). At its extreme carboxyl terminus, at the base of domain II, PBP 5 has a short 18-amino acid (Jackson & Pratt, 1987) amphi- pathic helix that tethers the protein to the outer face of the inner membrane (Nelson et al., 2002; Ghosh & Young, 2003). The closest homologue to PBP 5 from any organism is PBP 6, from E. coli itself. Interestingly, PBP 6, although 65% identical to PBP 5, cannot restore normal shape to aberrant cells, as can PBP 5 (Ghosh & Young, 2003). Domain swap and mutagenesis experiments indicate that the relevant differences between the two enzymes localize to domain I, and, in fact, to a small stretch of 20 amino acids that surrounds the canonical KTG motif of the active site (Nelson et al., 2002; Ghosh & Young, 2003). For FEMS Microbiol Lett 303 (2010) 76–83 c  2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved MICROBIOLOGY LETTERS Downloaded from https://academic.oup.com/femsle/article/303/1/76/559805 by guest on 05 November 2022