Studies on Enzymes of Prodigiosin Biosynthesis

Lead Research Organisation: University of Cambridge
Department Name: Chemistry

Abstract

Prodigiosin is a natural red tripyrrolic pigment with potent anti-cancer properties1. In a previous BBSRC-funded collaboration with Prof. George Salmond (Biochemistry), we elucidated the pathway for its biosynthesis and the enzymes responsible. One branch of the pathway makes a monopyrrole MAP and the steps are catalysed by the enzymes PigD, PigE, and PigB in that order. In this project we propose to study these three over-expressed and purified enzymes in vitro to verify that they do indeed catalyse the reactions proposed for them. PigD is proposed to be a TPP-dependent enzyme that transfers an acetyl group from pyruvate onto the B-position of an octenoyl derivative, probably a thioester, but the natural substrate has not been established. PigE seems to be a bifunctional enzyme that both reduces the thioester to an aldehyde and then performs a PLP-dependent transamination of the aldehyde. However the C-terminal half, proposed to be the reductase domain, is unlike any known reductase, so it will be particularly interesting to find out whether and how it catalyses the reduction. As well as studying its catalytic activity, we will try to obtain a crystal structure of PigE, not only because it is an entirely novel enzyme but also because their is an intriguing possibility that the reactive aldehyde intermediate gets channelled direct from one active site to the other without being released into solution. PigB is proposed to be a flavin-dependent oxidase that oxidises a dihydropyrrole to the pyrrole. We have previously synthesised the dihydropyrrole (and shown that it does restore prodigiosin biosynthesis to a mutant blocked earlier in the pathway2), however we were not able to effect the oxidation to a pyrrole by purely chemical means. Therefore PigB may be a useful (and cheap as it only uses O2) way to effect this valuable transformation. After establishing its activity in vitro we will study a range of substrate analogues to find out how broad a range substrates it can accept. We will also investigate whether the living bacterium can use these substrate analogues to make new analogues of prodigiosin.
This project will be an equal collaboration between Prof. Salmond and myself and requires skills from both groups, particularly molecular biology, genetic engineering and protein expression in Prof. Salmond's lab and synthesis and enzyme kinetics in my lab. It is therefore interdisciplinary. Prodigiosin's anti-cancer properties (not to mention the numerous other biological activities that have been reported) make it an important target. A close analogue of prodigiosin (obatoclax) has been put through Phase 2 clinical trials for treatment of several different types of cancer. Therefore the ability to make large quantities of either natural prodigiosin or closely related compounds by fermentation may be very valuable. To achieve this goal it is important that the activities of each enzyme are well understood. Therefore this study may contribute to both Industrial Biotechnology and Bioscience for Health.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011194/1 01/10/2015 30/09/2023
1645655 Studentship BB/M011194/1 01/10/2015 30/09/2019 Maxime Pierre Couturier
 
Description In prodigiosin biosynthesis, PigB catalyses the oxidation of dihydroMAP into the pyrrole MAP. We synthesised 10 new analogues of dihydroMAP with various substituents on the C2 an C3 positions. We then fed them into Serratia ?pigD, a mutant strain of Serratia sp. 39006 not able to produce dihydroMAP because PigD is not expressed. However, PigB is functional in that strain and the feeding of dihydroMAP or one of its analogues can lead to the recovery of prodigiosin production. We observed a recovery of pigmentation with 8 analogues: it seems that modification on the C3 positions were all accommodated, whereas only small chains (less than 4 carbons) could be accepted in the C2 position. This mutasynthesis led to 8 analogues of prodigiosin, including the first example with a bifurcated alkyl on the C3 position.
Serratia ?pigD was also fed with 3-acetyloctanal 1 and S-(2-acetamidoethyl) 3-acetyloctanethioate 2 and 3-acetyloctanoic acid 3. Recovery of pigmentation was observed with 1 and 2 but not with 3. The fact that 3 does not lead to any pigmentation means that even if 2 undergoes an hydrolysis in the media the resulting product cannot be accepted as a substrate in prodigiosin biosynthetic pathway. On the other hand, we were able to recover pigment when the bacteria were fed with 1 or 2. HP-LC analysis of the pigment obtained with 2 showed that it was indeed prodigiosin. This provess the presence of a thioester reductase in prodigiosin biosynthetic pathway and reinforce our hypothesis that PigE is a bifunctioannal enzyme, taking a thioester substrate, reuccing it to an aldehyde and finally transaminating it give the precursor of dihydroMAP.
Exploitation Route Prodigiosin displays several biological activity (anticancer and antiobiotic for instance). The mutasynthesis we have described is an aesy way to obtain a variety of prodigiosin analogue, whose activity could be promissing in term of drug discovery.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology