*Article* **Comparative Analysis, Structural Insights, and Substrate**/**Drug Interaction of CYP128A1 in** *Mycobacterium tuberculosis*

**Nokwanda Samantha Ngcobo 1, Zinhle Edith Chiliza 1, Wanping Chen 2, Jae-Hyuk Yu 3,4, David R. Nelson 5, Jack A. Tuszynski 6,7, Jordane Preto 8,\* and Khajamohiddin Syed 1,\***


Received: 29 April 2020; Accepted: 11 May 2020; Published: 8 July 2020

**Abstract:** Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms' primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing *Mycobacterium tuberculosis* H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different *in silico* approaches to understand CYP128 P450 family's distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 *CYP128* P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the *CYP128* gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between *M. tuberculosis complex* (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with *CYP124A1*, or together with *CYP124A1* and *CYP121A1*. The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in *M. tuberculosis*.

**Keywords:** cytochrome P450 monooxygenenases; CYP128A1; *Mycobacterium tuberculosis* H37Rv; tuberculosis; molecular dynamic simulations; azole drugs; menaquinone

#### **1. Introduction**

Tuberculosis (TB), caused by *Mycobacterium tuberculosis* H37Rv, remains a serious public health problem despite the existence of international TB control programs [1]. Recent data from the World Health Organization (WHO) shows that about 10 million people fell ill with TB in 2018 [1]. TB's global threat to human health has been exacerbated in recent years by the emergence of widespread multiand extensively drug-resistant *M. tuberculosis* strains [1]. The developing countries of South-East Asia and Africa have shown high incidence rates of TB. The prevalence of the disease in these countries is mainly due to lack of basic sanitation (causing an increase in transmission of the disease), human immunodeficiency virus (HIV) infection and lack of drugs to treat the disease [1]. Recent statistics from South Africa revealed that TB is the major killer among infectious diseases, indicating that this disease is still a major challenge in the country [2].

In 1998, determination of the *M. tuberculosis* H37Rv genome sequence encouraged more investigation of new anti-tubercular drugs and the seeking of more knowledge on the complex biology of the *M. tuberculosis* bacterium [3]. This highlighted the importance of lipid metabolism in *M. tuberculosis*; novel biosynthetic pathways were found to be involved in the synthesis of compounds such as phenolphthiocerol, mycolic acids and mycocerosic acid for the complex cell wall structure of the bacterium [4]. Among the enzymes involved in lipid metabolism, cytochrome P450 monooxygenases (CYPs/P450s) in *M. tuberculosis* were found to play a key role in the metabolism of lipids [5,6]. P450s are heme-thiolate proteins found in all species across biological domains [7]. Recent studies revealed the presence of a large number of P450s in mycobacterial species and most of these P450s were found to be involved in lipid metabolism [6]. *M. tuberculosis* H37Rv has 20 P450s in its genome and some of these P450s are indeed involved in lipid metabolism [5]. Furthermore, one of the P450 genes, namely *CYP125A1,* was used as a key factor in determining the cholesterol degrading ability of mycobacterial species [8].

Among *M. tuberculosis* H37Rv P450s, CYP128A1 gained particular interest among researchers owing to its history indicating its essentiality and its physiological importance. Transposon site hybridization mutagenesis studies indicated that *CYP128A1* is essential for in vitro survival of *M. tuberculosis* H37Rv [9]. Interestingly, another study, which used a similar approach, revealed that *CYP128A1* is not essential for survival of *M. tuberculosis* CDC1551 [10]. However, this study had a backdrop of limited gene coverage in its mutant library. In Vitro M. tuberculosis H37Rv latency model studies including a carbon starvation model [11] and hypoxia model [12] showed up-regulation of *CYP128A1*, suggesting that this P450 has a potential role in *M. tuberculosis* latency. Until 2016, the nature of *CYP128A1* with respect to its essentiality remained a mystery. Research revealed that *CYP128A1* is non-essential for survival of *M. tuberculosis* H37Rv as the *CYP128A1* gene knock-out strain survives [13]. However, the *CYP128A1* gene knock-out strain has proven to be hyper-virulent [13], indicating this gene actually playing a role in the synthesis of a compound that acts as a negative regulator of virulence.

Heterologous expression of *CYP128A1* posed a great challenge to researchers, as the expression of this particular P450 in *Escherichia coli* has been unsuccessful [14,15]. Genomic analysis revealed that *CYP128A1* is part of an operon that consists of two other genes, *stf3* and *rv2269c* [16]. In Vivo studies using *M. smegmatis* as model strain demonstrated that CYP128A1 is involved in hydroxylation of menaquinone-9 (MK9) and is essential in the synthesis of this compound, whereas Stf3 was found to introduce the sulfate group to menaquinone-9 and *rv2269c* was found to act as a promoter [13]. The sequence of reaction is that CYP128A1 introduces the hydroxyl group into MK9, followed by the addition of the sulfate group by the Stf3 that leads to the synthesis of sulfomenaquinone [13].

Lipoquinones are electron transport molecules that are involved in the respiratory function of bacteria and mainly consist of menaquinone and ubiquinone [17]. Menaquinones (2-methyl-3-polyprenyl-1,4-naphthoquinones) especially MK9 is ubiquitous and unique to mycobacteria [17], indicating that *CYP128A1* should be present in all mycobacterial species. However, to date, the distribution of *CYP128A1* in such a large number of mycobacterial species belonging to six

different mycobacterial categories, i.e., *Mycobacterium tuberculosis* complex (MTBC), *M. chelonae-abscessus* complex (MCAC), *M. avium* complex (MAC), mycobacteria causing leprosy (MCL), non-tuberculosis mycobacteria (NTM) and saprophytes (SAP) is still unknown. Anti-fungal azole drugs were shown to be promising new tools to fight TB, particularly as they showed high antimycobacterial activity [18–20] and interestingly, to date, characterized *M. tuberculosis* P450s have been found to bind quite a number of azole drugs [21], leading to *M. tuberculosis* P450s becoming the main focus as novel drug targets against this pathogen [5]. The binding of azole drugs to CYP128A1 could have an undesired effect, as this could lead to possible disruption of enzyme function, a vital component in the virulence modulation of *M. tuberculosis*.

To date, genome-wide analysis of *CYP128* P450s has only been carried out in 60 mycobacterial species [22] and because of the failure of *CYP128A1* heterologous expression [14,15], analysis of binding patterns of azole drugs to CYP128A1 has not been performed. To address these research gaps, in this study, genome-wide data mining, annotation, and phylogenetic analysis of CYP128A1 were carried out in 4250 mycobacterial species. Furthermore, *in silico* analysis of binding of different azole drugs with the CYP128A1 model was assessed. The results for CYP128A1 were discussed in the context of gaining more knowledge on the role of this P450 in mycobacterial species.
