Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis Stella A. Child a , Amna Ghith a , John B. Bruning b , Stephen G. Bell a,⁎ a Department of Chemistry, University of Adelaide, SA 5005, Australia b School of Biological Sciences, University of Adelaide, SA 5005, Australia ARTICLE INFO Keywords: Metalloenzymes Cholesterol Cytochrome P450 Mycobacterium X-ray crystallography Substrate binding ABSTRACT The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for or- ganism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tu- berculosis. The sequence of CYP125A7 from M.marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed diferences in the resting heme spin state and in the binding modes and afnities of certain azole inhibitors. CYP125A7 did not show a signifcant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some diferences in the extent of the Type I shifts to the high-spin form with steroids and a higher afnity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identifed as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria. 1. Introduction Mycobacterium tuberculosis is the pathogen responsible for human tuberculosis [1]. Tuberculosis continues to be a major human health concern, resulting in the deaths of 1.3 million people in 2017, and over ten million new infections [2]. The startling rise in multi-drug (MDR) and extensively-drug resistant (XDR) strains of this species makes tu- berculosis a growing, rather than diminishing, threat to human health despite a yearly decrease in incidence of 2% [2]. There is both a lack of efective new drugs on the market and difculties with existing treat- ment regimens (the standard treatment continues for six months), all of which increases the risk of substantial resistance developing, and em- phasises the necessity of further research into this bacterial species. Many other Mycobacterium species are also pathogenic; for example Mycobacterium leprae is the causal agent of leprosy [3]. Mycobacterium ulcerans infection results in the skin disease known as the Buruli or Daintree ulcer, which is common in Africa and increasingly so in northern and eastern Australia, and is the third most common human Mycobacterium pathogen after tuberculosis and leprosy [4,5]. Myco- bacterium marinum is a primarily a pathogen in frogs and fsh (although it can opportunistically infect humans, causing aquarium granuloma) [6]. It bears high sequence similarity to the human pathogens (85% and 97% to M. tuberculosis and M. ulcerans, respectively). This species is often used as a model organism for M. tuberculosis as the disease pro- gression and features (such as granuloma formation) are well conserved [7]. M. marinum has been hypothesised to resemble a common ancestor of the more pathogenic Mycobacterium species [6]. The non-pathogenic soil-dwelling bacterium Mycobacterium smegmatis is also sometimes used as a model species [8]. Cytochrome P450s (CYPs) are heme-thiolate monooxygenases, which perform the catalytic insertion of a single oxygen atom across a CeH bond. The sequencing of Mycobacterium genomes has revealed, among other things, that the number of cytochrome P450 genes in many Mycobacteria is unusually high for bacterial species [9,10]. In https://doi.org/10.1016/j.jinorgbio.2020.111116 Received 20 December 2019; Received in revised form 11 May 2020; Accepted 14 May 2020 ⁎ Corresponding author. E-mail address: stephen.bell@adelaide.edu.au (S.G. Bell). Journal of Inorganic Biochemistry 209 (2020) 111116 Available online 20 May 2020 0162-0134/ © 2020 Elsevier Inc. All rights reserved. T