Elucidating enzyme mechanism and physiological role of a key P450 enzyme (CYP121) from mycobacterium tuberculosis

Lead Research Organisation: University of Manchester
Department Name: Chem Eng and Analytical Science

Abstract

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Technical Summary

The structure of CYP121, a key cytochrome P450 enzyme in M. tuberculosis (Mtb), has been solved to 1 Angstrom at Leicester. We will capitalise on the UKs first P450 structure to establish physiological substrate and role, to reconstitute activity with the cloned reductase (FprA) and ferredoxin redox partners, and to address unresolved problems in P450 catalysis. These relate to proton delivery pathways and identification of catalytic intermediates that accumulate when proton relay is retarded. CYP121 is highly expressed in Mtb and production is regulated by cellular stresses. It is strongly inhibited by azole drugs, and their Kd values are paralleled by their efficiency as anti-mycobacterials. We will deconvulute the biology of this important pathogen P450 and analyse important structure/function questions that will advance our understanding of P450 mechanism.

Publications

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Balding PR (2008) How do azoles inhibit cytochrome P450 enzymes? A density functional study. in The journal of physical chemistry. A

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McLean KJ (2007) Structure, function and drug targeting in Mycobacterium tuberculosis cytochrome P450 systems. in Archives of biochemistry and biophysics

 
Description The highest quality (resolution) structure of a cytochrome P450 enzyme (CYP121 from the human pathogen Mycobacterium tuberculosis) was determined during the course of these studies. This enabled us to observe new properties of this enzyme, including its ability to bind heme in two orientations and the discovery of a pathway by which protons can be transferred within this enzyme to the heme group, and thus to allow this cytochrome P450 enzyme to split oxygen (O2) into its component atoms and to insert an oxygen atom into the substrate for this P450. The heme was also shown to have a distorted structure, which may underlie some of the unusual properties of the heme - including its difficult to reduce nature. The structure of CYP121 was also solved in complex with a tight binding inhibitor from the azole antifungal series (fluconazole). This inhbitor was shown to bind in an unusual mode by which a water molecule remains on the heme iron and is coordinated by a nitrogen atom from a fluoconazole triazole group. This results in a less extensive change in the heme absorbance properties than is seen when the this type of drug binds directly to the heme iron, and helps to explain the origin of similar observations made for binding of certain such drugs to other P450 enzymes. Protein partner systems able to pass electrons to the CYP121 were cloned/expressed from the M. tuberculosis bacterium, and non enzymatic routes to electron donation using laser based methods were also developed to provide tools to drive catalysis in CYP121, which we also showed was an essential gene for the viability of the pathogenic bacterium. Results reported relating to the effectiveness of azole antifungal drugs were reported widely in press and in online reports across the world.
Exploitation Route The structure of CYP121 provided the basis for work to make specific inhibitors of this essential enzyme for Mtb viability. This is ongoing in work with the group of Chris Abell in Cambridge using fragment screening. The substrate for CYP121 (the cyclic dipeptide cyclo-L-Tyr-L-Tyr) was identified by Belin et al, and it was found that CYP121 catalyzes a C-C bond coupling reaction to link the tyrosyl side chains. The function of this metabolite is unknown, but work to define its role might prove important in understanding Mtb infection and the reason for CYP121 gene essentiality. This work has also inspired us to form links with researchers (Dr. Claire Simons at Cardiff and Prof. Craig Hutton at Melbourne) who are making various analogues of cYY, as well as with Dr. Luiz de Carvalho at Mill Hill/The Crick Institute for studies of potency of new inhibitors against M tuberculosis. Some promising results have already been obtained. Various 2nd and 3rd generation molecules from fragment screening/linking/merging studies in collaboration with Chris Abell's group are now undergoing trials for their effectiveness against M. tuberculosis.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

URL http://www.manchester.ac.uk/discover/news/article/?id=2612
 
Description Our findings have led to ongoing work in our lab (and with UK and Australian collaborators) to generate specific inhibitors of the M. tuberculosis CYP121 protein, following the discovery that the enzyme is essential for viability of M. tuberculosis (through collaboration with Prof. Tanya Parish at QMUL/Seattle). A further study is ongoing in collaboration with the group of Chris Abell at Cambridge to develop specific CYP121 inhibitors using fragment based screening; and other efforts to characterize cYY substrate analogues as inhibitors of CYP121 are ongoing with groups in Melbourne and Cardiff. Other collaborative work with Dr. Luiz Carvalho at Mill Hill/Crick Institute is helping to define the potency of novel inhibitors against M. tuberculosis. Our work in this area continues and the key research done using funding from this grant has led to further collaboration with the Abell group (and others) that will lead to further publications in 2016.
First Year Of Impact 2012
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description BBSRC standard competitive grants scheme
Amount £522,461 (GBP)
Funding ID BB/I019227/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2012 
End 01/2015
 
Description Schools visit (Wilmslow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to primary school children in final year on general science/genetics - talk sparked questions and general discussion

Students registered interests in scientific career. Invite for further talk in following year obtained.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2014