Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.
The development of thermostable and solvent-tolerant metalloproteins is a long-sought goal for many applications in synthetic biology and biotechnology. In this work, we were able to engineer a highly thermostable and organic solvent-stable metallo variant of the B1 domain of protein G (GB1) with a tetrahedral zinc binding site reminiscent of the one of thermolysin. Promising candidates were designed computationally by applying a protocol based on classical and first-principles molecular dynamics simulations in combination with genetic algorithm optimization. The most promising of the computationally predicted mutants was expressed and structurally characterized and yielded a highly thermostable protein. The experimental results thus confirm the predictive power of the applied computational protein engineering approach for the de novo design of highly stable metalloproteins.
The intramolecular Diels-Alder reaction has been used as a powerful method to access the tricyclic core of the eunicellin natural products from a number of 9-membered-ring precursors. The endo/exo selectivity of this reaction can be controlled through a remarkable organocatalytic approach, employing MacMillan's imidazolidinone catalysts, although the mechanistic origin of this selectivity remains unclear. We present a combined experimental and density functional theory investigation, providing insight into the effects of medium-ring constraints on the organocatalyzed intramolecular Diels-Alder reaction to form the isobenzofuran core of the eunicellins.
Through millions of years of evolution, Nature has accomplished the development of highly efficient and sustainable processes and the idea to understand and copy natural strategies is therefore very appealing. However, in spite of intense experimental and computational research, it has turned out to be a difficult task to design efficient biomimetic systems. Here we discuss a novel strategy for the computational design of biomimetic compounds and processes that consists of i) target selection; ii) atomistic and electronic characterization of the wild type system and the biomimetic compounds; iii) identification of key descriptors through feature selection iv) choice of biomimetic template and v) efficient search of chemical and sequence space for optimization of the biomimetic system. As a proof-of-principles study, this general approach is illustrated for the computational design of a 'green' catalyst mimicking the action of the zinc metalloenzyme Human Carbonic Anhydrase (HCA). HCA is a natural model for CO2 fixation since the enzyme is able to convert CO2 into bicarbonate. Very recently, a weakly active HCA mimic based on a trihelical peptide bundle was synthetized. We have used quantum mechanical/molecular mechanical (QM/MM) Car-Parrinello simulations to study the mechanisms of action of HCA and its peptidic mimic and employed the obtained information to guide the design of improved biomimetic analogues. Applying a genetic algorithm based optimization procedure, we were able to re-engineer and optimize the biomimetic system towards its natural counter part. In a second example, we discuss a similar strategy for the design of biomimetic sensitizers for use in dye-sensitized solar cells.
Index S2 2D molecular structures S3 Global descriptors S7 Local descriptors S11 Comparison of global molecular descriptors S17 Correlation plot for chemical hardness S18 Comparison of local molecular descriptors S19 Efficacy of partial agonists S22 Cartesian coordinates of partial agonists (zwitterion)-B3LYP S23 Cartesian coordinates of partial agonists (zwitterion)-M06-2X S42 Cartesian coordinates of partial agonists (neutral)-B3LYP S48 Cartesian coordinates of antagonists-B3LYP S67 Cartesian coordinates of antagonists-M06-2X S91 Additional molecular structures-B3LYP S99 S3 2D molecular structures Table S1 Two-dimensional representations of selected antagonist molecules (*Yosa et al.[1]; **Di Fabio et al.[2]) ID 2D STRUCTURE pKi ID 2D STRUCTURE pKi 189* Table S11 Mulliken charge on the amide oxygen and nitrogen atom for the selected antagonists (M06-2X/6-311G**). ligand qO qN 189 -0.450 -0.564 192 -0.451 -0.556 210 -0.442 -0.560 213 -0.442 -0.561 217 -0.443 -0.560 29 -0.455 -0.564 41 -0.443 -0.575 24 -0.461 -0.560 38 -0.444 -0.544 44 -0.457 -0.563 Table S12 Mulliken charge on the amide oxygen and nitrogen atom for the selected partial agonists in their zwitterionic form (M06-2X/6-311G**). ligand qO qN Z9 -0.521 -0.474 Z15 -0.511 -0.481 Z19 -0.498 -0.467 Z22 -0.503 -0.468 Z26 -0.508 -0.472 Z29 -0.490 -0.466 Z35 -0.505 -0.468 Z39 -0.506 -0.468 Z46 -0.492 -0.460 Z51 -0.500 -0.470 S15 Table S13 Amide's carbonyl stretching frequency (cm -1 ), and isotropic magnetic chemical shielding constants for the amide's oxygen and nitrogen atom (ppm) for the antagonists (B3LYP/6-311G**).
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