Revelation of enzyme activity of mutant pyrazinamidases from Mycobacterium tuberculosis upon binding with various metals using quantum mechanical approach

Highlights

• Mutagenicity and metal substitution in PZase weakens the binding of PZA with PZase

• The present study aims at the quantum mechanistic analysis of mutant-metal substituted PZase complexes

• All the targeted mutations casts derogatory effect on the activity of PZase

• The substitution of iron with cobalt enhances the enzymatic activity of both wild type and mutant PZase

Abstract

Pyrazinamide (PZA) is one of the most potent bacteriostatic drug against tuberculosis, a deadliest disease with high mortality and morbidity rate. PZA metabolizes into its active form pyrazinoic acid (POA) with the help of a metalloenzyme, pyrazinamidase (PZase). Mutagenicity and metal substitution in PZase weakens the binding of PZA with PZase and increases the drug resistance in Mycobacterium tuberculosis. The present study aims at the quantum mechanistic analysis of mutant-metal substituted PZase complexes by studying the mechanics of metals and PZA binding at MCS and catalytic site, respectively. A total of 66 complexes are scrutinised in this study to elucidate the effect of mutations on the enzymatic function of PZase. Among the 10 mutations considered in this study, 7 different mutations i.e. Asp₄₉ → Asn, His₅₁ → Arg, Gly₇₈ → Cys, Asp₁₂ → Gly, Asp₁₂ → Ala, Thr₁₃₅ → Pro and Asp₁₃₆ → Gly cause a detrimental effect on the activity of PZase. In addition to this, the substitution of iron with cobalt enhances the enzymatic activity of both wild type and mutant PZase while zinc, magnesium and copper reduce it. Based on these results, it is concluded that upon substitution of iron with zinc, magnesium and copper, PZase cannot function properly. Due to mutations, the reactivity of the drug also reduces as its binding with PZase weakens and this phenomenon enhances the resistance of Mycobacterium tuberculosis against drug.

Introduction

Tuberculosis is one of the most lethal diseases in the world, as one-fourth of the global population is infected with this disease. In last year, around 1.7 million TB-related deaths have been reported worldwide (Stewart et al., 2018). Pyrazinamide (PZA), a potent drug against tuberculosis, is used as a short course therapeutic agent as it shortens the antituberculous therapeutic treatment time from 9 to 12 months to 6months. PZA has a strong potential against the persistence of tubercle bacilli, however, it works at an acidic pH after metabolizing into its active form i.e. pyrazinoic acid (POA) with the help of a metalloenzyme, Pyrazinamidase (PZase) (Saikia and Deka, 2014). PZase contains iron as a cofactor at its metal coordination site (MCS) and has an amidase activity (Heifets and Cangelosi, 1999; Saikia and Deka, 2014). At the MCS of PZase, iron binds with one aspartic acid residue and three histidine residues along with two water molecules as well (Harding et al., 2010). It has been previously reported that few metal ions could competitively bind with the PZase as a cofactor and would alter the structure and functionality of the PZase (Khadem-Maaref et al., 2017).

Furthermore, various mutations have been reported in PZase which makes the bacterium resistant to the therapeutic agents (Dooley et al., 2012; Juréen et al., 2008). Such mutations alter the enzymatic activity of PZase which results in lowering the efficacy of the drug. These mutations are very diverse in nature ranging from missense mutations to insertion/deletion mutations. Previous reports have elucidated that the binding of iron becomes weaker with few mutations occurring at MCS and this binding is crucial for the activity of PZase (Rasool et al., 2017; Sheen et al., 2012).

The computational analyses can help in analysing the effect of mutagenicity and binding of various metals with PZase on its enzyme functioning. After substituting other metals at MCS of mutant enzymes, the binding of the enzyme with PZA can be computationally determined by adapting the Density Functional Theory (DFT). DFT is a widely used computational quantum mechanics model for chemical and biological systems and works on the basis of various exchange-correlation functionals. Among all the functionals, B3LYP is a common hybrid exchange-correlation functional which is based on exact exchange from Hartree–Fock theory and other functional sources such as generalized gradient approximations from Becke88 and Lee-Yang-Parr (Minenkov et al., 2012; Orio et al., 2009).

Khadem-Maaref et al., 2017 elaborated the mode of binding of various metals at the MCS of PZase adapting the DFT exchange-correlation functionals. The results indicated that nickel and cobalt, both can restore the activity of PZase and can act as a cofactor instead of iron, effectively. Even nickel and cobalt are more active than iron, manganese and zinc for restoring the activity of wild-type PZase (Khadem-Maaref et al., 2017). Rasool et al. (2017) provided deep insights into the effect of mutagenicity on the activity of PZase based on DFT exchange-correlation functionals. The results suggested that the mutagenicity is deleterious for the PZase activity as it weakens the binding of the cofactor (iron) and prodrug (PZA), causing an increase in the drug-resistant nature of Mycobacterium Tuberculosis (Rasool et al., 2017).

In the present study, molecular level DFT based quantum mechanical analysis of mutant-metal substituted PZase complexes is performed for revealing the structural and mechanical properties of PZase, and their effect on the PZase activity. Primarily, the target of this study is to study the mechanics of metal and PZA binding at the MCS and catalytic site of PZase, respectively. The calculations are performed to elucidate the results of an experimental observation, previously reported by Sheen et al. (2012).