Identification of a novel carboxypeptidase encoded by Rv3627c that plays a potential role in mycobacteria morphology and cell division

Abstract Functionally uncharacterized gene Rv3627c is predicted to encode a carboxypeptidase in the pathogen of Mycobacterium tuberculosis (M. tuberculosis) , which remains a major threat to human health. Here, we sought to reveal the function of Rv3627c and to elucidate its effects on mycobacterial growth. Rv3627c was purified from E. coli using Ni 2+ -NTA affinity chromatography, and its identity was confirmed with a monoclonal anti-polyhistidine antibody. An enzyme activity assay involving a d -amino acid oxidase-peroxidase coupled colorimetric reaction and high-performance thin layer chromatography was performed. A pull-down assay and MS-MS were also employed to identify putative interaction partners of Rv3627c. Scanning electron microscopy and transmission electron microscopy were performed to observe any morphological alterations to Mycobacterium smegmatis (M. smegmatis) . We successfully obtained soluble expressed Rv3627c and identified it as carboxypeptidase using prepared peptidoglycan. Four proteins were identified as potential interaction partners with Rv3627c based on results obtained from both a pull-down assay and MS/MS analysis. Rv3627c over-expression induced M. smegmatis cells to become elongated, and promoted the formation of increased numbers of Z-rings. Rv3627c, a novel carboxypeptidase in M. tuberculosis identified in this study, exerts important effects on mycobacterial cell morphology and cell division. This functional information provides a promising insight into anti-mycobacterial target designs.