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

Jude Chenge,David Leys,Teresa Cortes,Max D Driscoll,Dijana Matak-Vinkovic,Kirsty J Mclean,Douglas B Young,Madeline E Kavanagh,Andrew W Munro,Colin W Levy,Stephen E J Rigby,Chris Abell

doi:10.1038/srep26628

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.

Highlights

Mycobacterium tuberculosis (Mtb) is the causative agent for tuberculosis (TB), a chronic, infectious human disease that is responsible for the death of more than 1.5 million people annually
A BLAST search using the Mtb CYP144A1 protein sequence revealed that CYP144 P450s are highly conserved within Mtb strains (~99–100% identity), and amongst the closely related Mtb complex (MTBC) members, which includes M. bovis (99%) and M. canettii (99%)
The majority of species containing CYP144A1 orthologues are directly associated with a human disease, including M. colombiense (64% identity to Mtb CYP144A1), a member of the MAC (Mycobacterium avium complex) which can infect human immune deficiency virus (HIV) patients with low CD4 cell counts[28,29,30,31]

Summary

Introduction

Mycobacterium tuberculosis (Mtb) is the causative agent for tuberculosis (TB), a chronic, infectious human disease that is responsible for the death of more than 1.5 million people annually. CYP51B1 is related to eukaryotic sterol 14α-demethylases (with fungal CYP51 enzymes being important targets for azole class drugs), while CYP121A1 is involved in secondary metabolite synthesis, catalysing the oxidative crosslinking of the aromatic side chains of the cyclic dipeptide cyclo-L-Tyr-L-Tyr (cYY) to form the product mycocyclosin. Mtb CYP144A1, encoded by the H37Rv gene Rv177722,23, was identified to bind tightly to several azole antifungals, raising the possibility that this P450 could be a target for novel anti-TB drug development. The evolutionary ancestry of CYP144A1 is explored, and its conservation across the Mycobacterium genus is consistent with its importance to Mtb. Data from transcriptome annotation are reported, which reveal that alternative transcripts of CYP144A1 are produced, leading to the production of different forms of the P450 protein. The successful crystallization of the shorter form of CYP144A1 (CYP144A1-TRV) and the first X-ray crystal structure of a CYP144A1 protein are reported, enabling novel insights into this enzyme, its active site organization and its relationship to other Mtb P450 enzymes

Methods

Results

Conclusion