Structural characterization of CYP144A1 – a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a “full-length” 434 amino acid version (CYP144A1-FLV) and (ii) a “truncated” 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5′-untranslated region and Shine-Dalgarno ribosome binding site.

In silico study of active compounds ADMET Profiling in Curcuma xanthorrhiza Roxb and Tamarindus indica as Tuberculosis Treatment

“…α-pinene could bind the 20 cytochrome P450 enzymes of Mycobacterium tuberculosis, involved in mycobacterial survival and pathogen in a human cell. Recently, it was reported that cyp144 and cyp130 encoded respectively by Rv1777 and Rv1256c could bind azole (Ouellet et al 2008, Chenge et al 2016, an antifungal drug that has anti-TB potent and it has been developing to be multidrug-resistant TB therapy (Gupta et al 2015). Isoamyl alcohol and furfural were showed that had interaction with alcohol dehydrogenase proteins.…”

Fragment Profiling Approach to Inhibitors of the Orphan M. tuberculosis P450 CYP144A1

“…All compounds except imidazole 4, clotrimazole, and econazole 18,24 produced heterogeneous sets of low spin g-values that indicate a minimum of two different coordination states of the heme iron. The implication that ligands might acquire different binding modes with respect to the CYP144A1 heme and that there remains a proportion of enzyme in the water-coordinated resting state when it is ligand bound prompted further investigation of the enzyme's ligand binding properties using competition NMR and UV−vis experiments.…”

“…The distinct binding modes of compounds 1−3 to CYP144A1 and CYP121A1 was rationalized based on the chemical properties and steric constraints imposed by the proximal active site residues of CYP144A1 and the hydrophobicity of the CYP144A1 distal active site. 24 The ligand profile and the biophysical characteristics of CYP144A1, elucidated using these novel ligands, provide insight into the potential biological function of the enzyme. The predicted conformational flexibility and the open structure of the CYP144A1 active site, in addition to the low susceptibility of the protein to inhibition with a range of common heme binding functional groups, could indicate a role for CYP144 in xenobiotic detoxification.…”

“…All ligand binding experiments that are reported were performed using a His 6 -tagged construct of the truncated version of the CYP144A1 protein (CYP144A1-TRV) . No significant differences were observed for ligand binding experiments conducted using the full length CYP144A1 protein and both versions of the CYP144A1 enzyme have previously been shown to have near identical thermal stability, optical and electronic properties. , All reagents and fragments were obtained from commercial sources. Aminopyrazole CYP121A1 inhibitors were synthesized according to previously reported procedures .…”

Potential drug targets in the Mycobacterium tuberculosis cytochrome P450 system

“…However, a similar finding was observed when CYP125A1 was knocked out (56). More detailed analysis revealed that there are two possible start codons for CYP144A1, both of which give rise to proteins that can be expressed and purified (21). The terminal region of the longer protein appears to be disordered, but the truncated, shorter protein was amenable to crystallization and its ligand-free structure was determined.…”