RRC ID 60636
Author Saroja NR, Mohan AHS, Srividya D, Supreetha K.
Title Chaperone-assisted expression and purification of putrescine monooxygenase from Shewanella putrefaciens-95.
Journal Protein Expr Purif
Abstract A putrescine monooxygenase from Shewanella putrefaciens 95 (SpPMO) is the initial enzyme catalyzing the hydroxylation of putrescine to N-hydroxyl putrescine, the precursor for the synthesis of a siderophore putrebactin was identified. This PMO clustered together with known characterized NMOs from Shewanella baltica, Bordetella pertussis, Erwinia amylovora, Streptomyces sp. Gordonia rubripertincta, Pseudomonas aeruginosa and outgrouped from Escherichia coli, Nocardia farcinica, and Rhodococcus erythropolis. The deduced SpPMO protein showed 53% and 36% sequence identity with other characterized bacterial NMOs from Erwinia amylovora and Gordonia rubripertincta respectively. In this investigation, we have cloned the complete 1518bp coding sequence of pubA from Shewanella putrefaciens 95 encoding the corresponding protein SpPMO. It comprises 505 amino acid residues in length and has approximately a molecular weight of 54 kDa. Chaperone-assisted heterologous expression of SpPMO in pET151Topo expression vector under the control of bacteriophage T7 promoter permitted a stringent IPTG dependent expression. It has been successfully cloned, overexpressed and purified as a soluble His6 -tagged enzyme using E. coli as a cloning and expression host. The expression of recombinant SpPMO was confirmed by Western blotting using anti-His6 antibody. The purified protein showed FAD and NADPH dependent N-hydroxylation activity. This study has paved a way to understand the hydroxylation step of putrebactin synthesis which can be further investigated by studying its kinetic mechanism and physiological role.
Volume 157
Pages 9-16
Published 2019-5-1
DOI 10.1016/j.pep.2019.01.006
PII S1046-5928(18)30539-4
PMID 30654014
MeSH Amino Acid Sequence Bacterial Proteins / chemistry Bacterial Proteins / genetics* Bacterial Proteins / isolation & purification Bacterial Proteins / metabolism Cloning, Molecular / methods* Escherichia coli / genetics Flavin-Adenine Dinucleotide / metabolism Hydroxylation Mixed Function Oxygenases / chemistry Mixed Function Oxygenases / genetics* Mixed Function Oxygenases / isolation & purification Mixed Function Oxygenases / metabolism Molecular Chaperones / genetics Molecular Chaperones / metabolism NADP / metabolism Putrescine / metabolism Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Recombinant Proteins / metabolism Sequence Alignment Shewanella putrefaciens / chemistry Shewanella putrefaciens / genetics* Shewanella putrefaciens / metabolism
IF 1.291
Times Cited 1
Resource
General Microbes JCM20190