A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.
DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Ankita
A
Bansal
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Debasish
D
Kar
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Rajagopal A
RA
Murugan
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Sathi
S
Mallick
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Mouparna
M
Dutta
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Satya Deo
SD
Pandey
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Chiranjit
C
Chowdhury
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal PIN-721302, India.
Anindya S
AS
Ghosh
D000107:Acetylation; D020816:Amino Acid Motifs; D017124:Conserved Sequence; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D005816:Genetic Complementation Test; D006868:Hydrolysis; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D013379:Substrate Specificity; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism / Q000494:pharmacology
D046915:Penicillin-Binding Proteins; D010406:Penicillins; D047090:beta-Lactams; D004150:Dipeptidases; C019989:DD-carboxypeptidase-endopeptidase; D001618:beta-Lactamases