Angad Mehta
Departmental Seminar Title: Mechanistic Insights in the biosynthesis of Pyoluteorin
Semiar date: 18th Oct, 2010.
Abstact:
Several medically and agriculturally important natural products contain pyrrole moieties;
pyoluteorin (Plt) is one of them. It is an antifungal agent produced by fluorescent Pseudomonads
found in the rhizosphere of plants. The biosynthesis of Plt involves novel pyrrole biosynthesis. It
involves an L-prolyl-AMP ligase which activates L-proline for further thioestrification to form
prolyl-S-peptidyl carrier protein (prolyl-S-PCP). Prolyl-S-PCP then undergoes four electron
oxidation to form Pyrrolyl-S-PCP. The pyrrole derivative so formed undergoes flavin dependent
dichlorination to form 4,5-dichloropyrrole moiety. This chlorination step is characterized in terms
of localization of dichlorination, time course for the formation of dichloropyrrole and halogenations
using bromide instead of chloride. The biosynthesis of prolyl-S-PCP as well as the downstream
halogenation steps have been studied by tandem mass spectrometry. The next step in the
biosynthesis involves biosynthesis of resorcinol followed by its coupling to dichlorinated pyrrole
moiety. This part of biosynthesis of Plt is still unexplored. This review revisits mechanistic insights
into the biosynthesis of dichlorinated pyrrole moiety followed by a proposed mechanism for
biosynthesis of resorcinol ring and its coupling to dichlorinated pyrrole to form Plt.
Introduction:
Fluorescent Pseudomonads are commonly found in the rhizosphere of plants. These Pseudomonads
are known to suppress soil borne fungal pathogens by producing antifungal agents. The inhibition
of most of the fungal diseases essentially depends on their biosynthesis and secretion of these
secondary metabolites. Among these exo-products antibiotics have proved to be effective agents for
their biocontrol properties.1,2,3. Pyrrolnitrin, phenazine, 2,4-diacetylphloroglucinol, pyoluteorin (Plt)
are the common antibiotics produced by fluorescent pseudomonads. This review includes
mechanistic insights in the biosynthesis of Plt.
Biosynthetic gene cluster and Proposed biosynthetic pathway for Plt:2,4
The Plt biosynthetic gene cluster has ten genes pltABCDEFG and regulators pltR and pltZ. PltB/C
are annotated as type I polyketide synthase, pltF is a putative amino acid-AMP ligase, pltL is a
putative peptidyl carrier protein, pltA/D/M are annotated as flavin dependent halogenases, pltG is
annotated as thioesterase and pltE as acyl-CoA dehydrogenase.
Based on these annotations the proposed biosynthetic pathway is as shown below:
Fig.1: Proposed biosynthetic pathway.
Biosynthesis of Pyrrole moiety:
The enzymes involved in the formation of the pyrrole moiety are PltF, PltL and PltE. From
precursor labeling studies it was found that L-proline was the biosynthetic precursor for the
formation of pyrrole. This led to the characterization of the enzymes in this pathway. PltF was
characterized as L-prolyl-AMP ligase which activates L-proline for further attack by the thiol
moiety of 4’-phosphopantheine attached to peptidyl carrier protein to generate prolyl-S-PCP
intermediate. PltE, the putative acyl-CoA dehydrogenase, then does a FAD mediated two electon
oxidation to form FADH2 and pyrrolinyl-2-caboxyl-S-PCP which can further undergo enzymatic or
non-enzymatic 2 electron oxidation to form pyrrolyl-2-carboxyl-S-PCP. The products of
reconstitution of the pyrrole moiety have been characterized by NMR and tandem mass
spectrometry techniques.5
Fig.2 Formation of pyrrolyl-2-carboxy-S-PCP
Dichorination of pyrrolyl-S-carrier Protein:
There are more than 4000 chlorinated natural products identified. The deschloro analogs of many of
these natural products are less biologically active. This is true for Plt as well where the deschloro
analogue is 8 fold less active against Bacillus subtilis. PltA/M/D were putative halogenases.
However, PltD lacks FAD binding motif and may be non functional. PltA is an active halogenase
and does dihalogenation and PltM cannot substitute the activity for PltA.It also does not
complement the activity of PltM. PltA is a FAD dependent halogenase. This chlorination step is
characterized in terms of localization of dichlorination, time course for the formation of
dichloropyrrole and halogenations using bromide instead of chloride by Tandem mass
spectrometry.6
C
Fig.3 (a) and (b) indicate the formation of mochlorinated product vs dichlorinated product. (c)
indicates that PltA is the active halogenase and PltM has no effect on chlorination.
Fig.4 Proposed mechanism for chlorination.
Proposed biosynthesis of the resorcinol Moiety:
PltB and PltC are annotated as type I polyketide synthase. Also, PltG is annotated as a thioesterase.
Hence it can be speculated that pltG can be involved in the transfer of the dichlorinated pyrrole
moiety from PltL to the first module of PltB where it can undergo carbon chain extension using
three malonyl CoA units. Resorcinol ring may be formed via condensation reaction followed by
dehydration and tautomerization to give Plt. This is illustrated in the figure below.
Conclusion:
The biosynthesis of the pyrrole moiety of Plt is thoroughly characterized. However, the exact
mechanism of chlorination is yet unknown. Some studies on halogenase speculate a generation of
HOCL species which then does an electrophilic halogenation.8 Also, the polyketide synthease
involved in biosynthesis of Plt are yet to be characterized. Characterization of these steps may be
useful for unlocking the potential for natural product diversification so that the analogs of these
compounds can be used as herbicide, antifungal agents and pesticides.
References:
1. Yi-He Ge, Dong-Li Pei, Yan-Hong Zhao, Wei-Wei Li, Shu-Fang Wang, Yu-Quan Xu,
Current Microbilogy, 54, 2007, 277
2. Jennifer Kraus, Joyce Loper, Applied and Environmental Microbiology, 1995, 849-854
3. Xianqing Huang, Xuehong Zhang, Yuquan Xu, Research in Mucrobiology, 159, 2008,
128-136.
4. Brian Nowak-Thompson, S. Gould, Joyce Loper, Gene, 204, 1997, 17-24.
5. Michael Thomas, Michael Burkart, Christopher Walsh, Chemistry and Biology, 9, 171184
6. Dorrestein, P.C., Ellen yeh, Gernau-Tsodikova, S., Neil Kelleher, Christopher T. Walsh,
PNAS, 102, 39, 13843-13848.
7. Brian Nowak-Thompson, S. Gould, Joyce Loper, Gene, 204, 1997, 17-24
8. Ellen Yeh, Leah C. Blasiak, Alexander Koglin, Catherine L. Drennan, and Christopher
T. Walsh, Biochemistry, 2007, 46, 1284-1292.