Report On The Putrescine Monooxygenase From Water Dwelling Shewanella Putrefaciens 95
The putrescine monooxygenase from fresh water/ sea water dwelling Shewanella putrefaciens 95 was investigated with respect to cloning, expression and purification. For the first time it is shown that the Shewanella genus had a gene cluster that produced putrebactin. Putrescine monooxygenase (PMO), Succinyl coA transferase and oligomerization enzymes are the three enzymes encoded by pubA, pub and pubC. As PMO is the first enzyme to create the iron chelating organic molecule, it can be harnessed as a drug target for bacterial diseases and this the main objective of our investigation. With the availability of genome sequence of Shewanella putrefaciens 200, the retrieval of PMO sequence was possible concluding that a similar gene cluster exists in S. putrefaciens 95 and S. purefaciens 200. The putative putrescine N-hydroxylating monooxygenase was amplified and the coding sequence was deposited with the accession number MH899123. The BLAST search showed with SpPMO shared a high sequence similarity with Shewanella putrefaciens 200 and Shewanella baltica. Next identity was shared with NMOs from Bordetella pertusis, Erwinia amylovora and Streptomyces spp involved in the alcaligin and desferrioxamine synthesis. Bioinformatic analysis showed that superimposition of 3D structure of cadeverine monooxygenase (DfoA) from Erwinia amylovora on SPPMO was near to 95% and revealed that tertiary structure of the two proteins is highly similar.
Interesting information is that in the phylogenetic tree the outgroups for SpPMO are putreescine oxidase from Rhodococcus erythropolis, though functioning similar to monooxygenase.The most successfully studied FMOs are the anzymes that have been produced in the soluble form and later purified and crystallized. His tagged purification has been a choice for studying these and there are bottlenecks even today in getting the proteins in soluble form. This study demonstrated that SpPMO is purified with intact enzymatic properties though the protein concentration of the purified enzyme is moderate. One of the factors that would have played in getting a soluble form of this enzyme would be E. coli chaperones Gro ES/EL. It is known that these bacterial chaperones play a vital role in folding and assembly of proteins and is also known to impart functionality to metalloenzymes.
In conclusion, SpPMO is an enzyme that catalyses the O2 and FADH2 dependent hydroxylation of putrescine to give N- hydroxyputrescine. It was purified to homogeneity from siderophore producing microorganism Shewanella putrefaciens 95 strain. SpPMO is a monomer, contains FAD as a prosthetic group, with NADPH as a cofactor, with high affinity towards putrescine and lysine as substrates. Optimization of the native folding of recombinant SpPMO, formulated a simple protocol that combined chaperon assistance during recombinant protein expression. This provided us an input to solubilize the protein of interest. Even though FAD enzymes are challenging to express in active form, with modifications in cloning stategy and purification process, we showed that SpPMO belonging to class B monooxygenases can be expressed and purified in active form.
To favor further de novo native folding fusion tags like MBP, creating clones with truncated version of gene or mutating SpPMO gene at suitable regions,(ref) changing the host of expression could be explored for better expression and easy purification. Future work would be studying the mechanism of catalysis and crystallization of SpPMO.