SwePub - sökning: WFRF:(John Juliane 1987 )

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1.	Lebrette, Hugo, 1986-, et al. (författare) Structure of a ribonucleotide reductase R2 protein radical 2023 Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 382:6666, s. 109-113 Tidskriftsartikel (refereegranskat)abstract Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.
2.	John, Juliane, 1987- (författare) High (valent) on O2 : Ribonucleotide Reductase and Methane Monooxygenase 2023 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Macromolecular X-ray crystallography (MX) is a powerful method to investigate protein structures. However, proteins with redox-active centres and radicals are very susceptible to photoreduction. It is therefore challenging to acquire structural details of redox-active centres in defined oxidation states or protein radicals using synchrotron radiation. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) radiation mitigates this problem. XFELs produce intense pulses of femtosecond length that give rise to diffraction before photoinduced movement can occur in the illuminated protein. Additionally, SFX allows experiments at room temperature and induction of reactions in crystallo. In this thesis two different redox-active enzyme systems were investigated with MX and SFX. The first part examines ribonucleotide reductase (RNR). RNR is the only known enzyme to synthesize de novo deoxyribonucleotides, the building blocks of DNA. Class I RNR consists of a small subunit R2 and a large subunit R1. R2 generates a radical in an oxygen dependent way and delivers it to R1 for ribonucleotide reduction. After catalysis the radical is transferred back to R2 until further use. Class I RNR is divided in five subclasses, mostly based on their mechanism of radical generation. In Paper I class Ib R2 is investigated. R2b binds two manganese ions that react with superoxide to produce a radical. The superoxide is provided by a small flavoprotein, NrdI, bound to R2. When exposed to molecular oxygen, reduced NrdI generates superoxide that is transferred to the R2 active site. Here two SFX structures of reduced and oxidized NrdI in complex with R2 are presented and it is suggested how the binding and NrdI oxidation state could influence the superoxide production. In Paper II the SFX structure of a R2e protein radical is presented. Class Ie R2 contains a metal-free active site. The comparison of the radical structure with a ground state structure highlights the changes induced by radical formation. A mechanism for the initiation of the radical transfer to R1 is proposed based on the structural details observed. In Paper III light is shed on a new variant of R2e. Three of the typically conserved active site residues are mutated in R2e; from three glutamates to valine, proline and lysine (VPK) or to glutamine, serine and lysine (QSK). Other publications, including Paper II, describe the VPK mutation but as of now the QSK variant has not been examined. Here, crystal structures of a R2e QSK protein are shown. A tyrosine close to the active site is post-translationally modified to a dihydroxyphenylalanine (DOPA). The amount of modified protein is shown to scale with the coexpression of other proteins of the RNR operon. The second redox-active enzyme investigated is soluble methane monooxygenase (sMMO). sMMO oxidizes methane to methanol and is produced by methanotrophs; bacteria that use methane as their sole carbon source. Methane is a potent greenhouse gas and can be found in ever increasing concentrations in the atmosphere due to human activities; sMMO is thus a compelling target for biotechnological development. Paper IV presents SFX structures of the catalytic subunit MMOH in complex with its small regulatory subunit MMOB in the oxidized and reduced resting state. It is also demonstrated that the complex can undergo the catalytic cycle in crystallo, allowing investigation of reaction cycle intermediates in the future.

John, Juliane, 1987- (författare)
High (valent) on O2 : Ribonucleotide Reductase and Methane Monooxygenase
2023
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Macromolecular X-ray crystallography (MX) is a powerful method to investigate protein structures. However, proteins with redox-active centres and radicals are very susceptible to photoreduction. It is therefore challenging to acquire structural details of redox-active centres in defined oxidation states or protein radicals using synchrotron radiation. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) radiation mitigates this problem. XFELs produce intense pulses of femtosecond length that give rise to diffraction before photoinduced movement can occur in the illuminated protein. Additionally, SFX allows experiments at room temperature and induction of reactions in crystallo. In this thesis two different redox-active enzyme systems were investigated with MX and SFX. The first part examines ribonucleotide reductase (RNR). RNR is the only known enzyme to synthesize de novo deoxyribonucleotides, the building blocks of DNA. Class I RNR consists of a small subunit R2 and a large subunit R1. R2 generates a radical in an oxygen dependent way and delivers it to R1 for ribonucleotide reduction. After catalysis the radical is transferred back to R2 until further use. Class I RNR is divided in five subclasses, mostly based on their mechanism of radical generation. In Paper I class Ib R2 is investigated. R2b binds two manganese ions that react with superoxide to produce a radical. The superoxide is provided by a small flavoprotein, NrdI, bound to R2. When exposed to molecular oxygen, reduced NrdI generates superoxide that is transferred to the R2 active site. Here two SFX structures of reduced and oxidized NrdI in complex with R2 are presented and it is suggested how the binding and NrdI oxidation state could influence the superoxide production. In Paper II the SFX structure of a R2e protein radical is presented. Class Ie R2 contains a metal-free active site. The comparison of the radical structure with a ground state structure highlights the changes induced by radical formation. A mechanism for the initiation of the radical transfer to R1 is proposed based on the structural details observed. In Paper III light is shed on a new variant of R2e. Three of the typically conserved active site residues are mutated in R2e; from three glutamates to valine, proline and lysine (VPK) or to glutamine, serine and lysine (QSK). Other publications, including Paper II, describe the VPK mutation but as of now the QSK variant has not been examined. Here, crystal structures of a R2e QSK protein are shown. A tyrosine close to the active site is post-translationally modified to a dihydroxyphenylalanine (DOPA). The amount of modified protein is shown to scale with the coexpression of other proteins of the RNR operon. The second redox-active enzyme investigated is soluble methane monooxygenase (sMMO). sMMO oxidizes methane to methanol and is produced by methanotrophs; bacteria that use methane as their sole carbon source. Methane is a potent greenhouse gas and can be found in ever increasing concentrations in the atmosphere due to human activities; sMMO is thus a compelling target for biotechnological development. Paper IV presents SFX structures of the catalytic subunit MMOH in complex with its small regulatory subunit MMOB in the oxidized and reduced resting state. It is also demonstrated that the complex can undergo the catalytic cycle in crystallo, allowing investigation of reaction cycle intermediates in the future.

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