A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis

• Published in: The Protein Journal Vol. 36; no. 3; pp. 220 - 227
• Main Authors: Bansal, Ankita, Kar, Debasish, Pandey, Satya Deo, Matcha, Ashok, Kumar, N. Ganesh, Nathan, Soshina, Ghosh, Anindya S.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.06.2017; Springer; Springer Nature B.V
• Subjects: Alanine; Analysis; Animal Anatomy; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Beta lactamases; beta-Lactamases - chemistry; beta-Lactamases - genetics; Biochemistry; Bioorganic Chemistry; Carbenicillin; Chemistry; Chemistry and Materials Science; Drug resistance in microorganisms; E coli; Enzymatic activity; Escherichia coli; Glutamate; Glutamic acid; Glutamic Acid - chemistry; Glutamic Acid - genetics; Histology; Hydrogen bonds; Hydrolysis; Minimum inhibitory concentration; Morphology; Mycobacterium smegmatis - enzymology; Mycobacterium smegmatis - genetics; Organic Chemistry; Penicillin; Phenols; Phenylalanine; Protein Structure, Secondary; Structure-function relationships; Ticarcillin; Tyrosine; Tyrosine - chemistry; Tyrosine - genetics; Beta-lactamase; Mycobacterium; Site-directed mutagenesis; Penicillinase
• ISSN: 1572-3887; 1573-4943; 1875-8355
• DOI: 10.1007/s10930-017-9713-2

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure–function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.