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X-ray structure of a Hg2+ complex of mercuric reductase (MerA) and quantum mechanical/molecular mechanical study of Hg2+ transfer between the C-terminal and buried catalytic site cysteine pairs.

ABSTRACT: Mercuric reductase, MerA, is a key enzyme in bacterial mercury resistance. This homodimeric enzyme captures and reduces toxic Hg2+ to Hg0, which is relatively unreactive and can exit the cell passively. Prior to reduction, the Hg2+ is transferred from a pair of cysteines (C558' and C559' using Tn501 numbering) at the C-terminus of one monomer to another pair of cysteines (C136 and C141) in the catalytic site of the other monomer. Here, we present the X-ray structure of the C-terminal Hg2+ complex of the C136A/C141A double mutant of the Tn501 MerA catalytic core and explore the molecular mechanism of this Hg transfer with quantum mechanical/molecular mechanical (QM/MM) calculations. The transfer is found to be nearly thermoneutral and to pass through a stable tricoordinated intermediate that is marginally less stable than the two end states. For the overall process, Hg2+ is always paired with at least two thiolates and thus is present at both the C-terminal and catalytic binding sites as a neutral complex. Prior to Hg2+ transfer, C141 is negatively charged. As Hg2+ is transferred into the catalytic site, a proton is transferred from C136 to C559' while C558' becomes negatively charged, resulting in the net transfer of a negative charge over a distance of ?7.5 Å. Thus, the transport of this soft divalent cation is made energetically feasible by pairing a competition between multiple Cys thiols and/or thiolates for Hg2+ with a competition between the Hg2+ and protons for the thiolates.

SUBMITTER: Lian P

PROVIDER: S-EPMC4245977 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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X-ray structure of a Hg2+ complex of mercuric reductase (MerA) and quantum mechanical/molecular mechanical study of Hg2+ transfer between the C-terminal and buried catalytic site cysteine pairs.

Lian Peng P Guo Hao-Bo HB Riccardi Demian D Dong Aiping A Parks Jerry M JM Xu Qin Q Pai Emil F EF Miller Susan M SM Wei Dong-Qing DQ Smith Jeremy C JC Guo Hong H

Biochemistry 20141113 46

Mercuric reductase, MerA, is a key enzyme in bacterial mercury resistance. This homodimeric enzyme captures and reduces toxic Hg2+ to Hg0, which is relatively unreactive and can exit the cell passively. Prior to reduction, the Hg2+ is transferred from a pair of cysteines (C558' and C559' using Tn501 numbering) at the C-terminus of one monomer to another pair of cysteines (C136 and C141) in the catalytic site of the other monomer. Here, we present the X-ray structure of the C-terminal Hg2+ comple ...[more]

PMID: 25343681

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Project description:High-order quantum chemistry is applied to hydrogen-bonded natural DNA nucleobase pairs [adenine:thymine (A:T) and guanine:cytosine (G:C)] and non-natural Hachimoji nucleobase pairs [isoguanine:1-methylcytosine (B:S) and 2-aminoimidazo[1,2a][1,3,5]triazin-4(1H)-one:6-amino-5-nitropyridin-2-one (P:Z)] to see how the intermolecular interaction energies and their energetic components (electrostatics, exchange-repulsion, induction/polarization, and London dispersion interactions) vary among the base pairs. We examined the Hoogsteen (HG) geometries in addition to the traditional Watson-Crick (WC) geometries. Coupled-cluster theory through perturbative triples [CCSD(T)] extrapolated to the complete basis set (CBS) limit and high-order symmetry-adapted perturbation theory (SAPT) at the SAPT2+(3)(CCD)δMP2/aug-cc-pVTZ level are used to estimate highly accurate noncovalent interaction energies. Electrostatic interactions are the most attractive component of the interaction energies, but the sum of induction/polarization and London dispersion is nearly as large, for all base pairs and geometries considered. Interestingly, the non-natural Hachimoji base pairs interact more strongly than the corresponding natural base pairs, by -21.8 (B:S) and -0.3 (P:Z) kcal mol-1 in the WC geometries, according to CCSD(T)/CBS. This is consistent with the H-bond distances being generally shorter in the non-natural base pairs. The natural base pairs are energetically more stabilized in their Hoogsteen geometries than in their WC geometries. The Hoogsteen geometry makes the A:T base pair slightly more stable, by -0.8 kcal mol-1, and it greatly stabilizes the G:C+ base pair, by -15.3 kcal mol-1. The G:C+ stabilization is mainly due to the fact that C has typically added a proton when found in Hoogsteen geometries. By contrast, Hoogsteen geometries are substantially less favorable than WC geometries for non-natural Hachimoji base pairs, by 17.3 (B:S) and 13.8 (P:Z) kcal mol-1.

Project description:The hypersaline Kebrit Deep brine pool in the Red Sea is characterized by high levels of toxic heavy metals. Here, we describe two structurally related mercuric reductases (MerAs) from this site which were expressed in Escherichia coli Sequence similarities suggest that both genes are derived from proteobacteria, most likely the Betaproteobacteria or Gammaproteobacteria We show that one of the enzymes (K35NH) is strongly inhibited by NaCl, while the other (K09H) is activated in a NaCl-dependent manner. We infer from this difference that the two forms might support the detoxification of mercury in bacterial microorganisms that employ the compatible solutes and salt-in strategies, respectively. Three-dimensional structure modeling shows that all amino acid substitutions unique to each type are located outside the domain responsible for formation of the active MerA homodimer, and the vast majority of these are found on the surface of the molecule. Moreover, K09H exhibits the predominance of acidic over hydrophobic side chains that is typical of halophilic salt-dependent proteins. These findings enhance our understanding of how selection pressures imposed by two environmental stressors have endowed MerA enzymes with catalytic properties that can potentially function in microorganisms that utilize distinct mechanisms for osmotic balance in hypersaline environments.IMPORTANCE Analysis of two structurally homologous but catalytically distinct mercuric reductases from the Kebrit Deep brine in the Red Sea sheds light on the adaptations that enable microorganisms to cope simultaneously with extreme salinity and toxic mercury compounds. One is strongly inhibited by high NaCl concentrations, while the other exhibits NaCl-dependent activation. Their different activity profiles imply that they may derive from bacterial microorganisms that utilize compatible solutes and salt-in strategies, respectively, to maintain osmotic balance. Three-dimensional modeling reveals that regions not involved in formation of the active homodimer are conserved between the two. However, in the NaCl-dependent form, distinct amino acid substitutions are found in areas that are critical for stability in high salt. The work provides insights into how two environmental stressors have shaped the structure of orthologous enzymes through selection and adaptation, enabling them to retain their catalytic function in what may be very different cellular contexts.

Dataset Information

X-ray structure of a Hg2+ complex of mercuric reductase (MerA) and quantum mechanical/molecular mechanical study of Hg2+ transfer between the C-terminal and buried catalytic site cysteine pairs.

Publications

X-ray structure of a Hg2+ complex of mercuric reductase (MerA) and quantum mechanical/molecular mechanical study of Hg2+ transfer between the C-terminal and buried catalytic site cysteine pairs.

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