Ultrafast catalytic processes and conformational changes in the light-driven enzyme protochlorophyllide oxidoreductase (POR)

Sytina, Olga A.; Heyes, Derren J.; Hunter, C. Neil; Groot, Marie Louise

doi:10.1042/bst0370387

Cited by 19 publications

(17 citation statements)

References 41 publications

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“…LPOR and DPOR [23], [25], [31]), other steps of this complex pathway have only been elucidated by gene knockouts/deletions, complementation, and mutational studies [14], [32], [33]. Many of these enzymes form complexes, catalyze novel reactions and may interact with yet to be identified protein partners [34], making biochemical studies as well as heterologous pathway reconstitution particularly challenging.…”

Section: Introductionmentioning

confidence: 99%

A Tale of Two Reductases: Extending the Bacteriochlorophyll Biosynthetic Pathway in E. coli

Tikh

Quin

2014

PLoS ONE

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The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in E. coli. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms Rhodobacter sphaeroides and Chlorobaculum tepidum. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg2+ in the porphyrin ring, with no activity against non-Mg2+ chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both in vitro, and in vivo as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in E. coli. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.

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Section: Introductionmentioning

confidence: 99%

A Tale of Two Reductases: Extending the Bacteriochlorophyll Biosynthetic Pathway in E. coli

Tikh

Quin

2014

PLoS ONE

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“…In this regard, it was recently shown that a conformational change at the active site of the POR enzyme is necessary to activate catalysis. This first reaction step is then followed by the actual photochemical reaction, which results in the formation of a reaction intermediate (I 675 *) with rate constants of 3 and 100 ns À1 (11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

Excited-State Dynamics of Protochlorophyllide Revealed by Subpicosecond Infrared Spectroscopy

Colindres-Rojas

Wolf

Gross

et al. 2011

Biophysical Journal

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To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ₁ = 3.6 ± 0.2, τ₂ = 38 ± 2, and τ₃ = 215 ± 8 ps. As indicated by the C13'=O carbonyl stretching mode in the electronic ground state at 1686 cm⁻¹, showing partial ground-state recovery, and in the excited electronic state at 1625 cm⁻¹, showing excited-state decay, τ₂ describes the formation of a state with a strong change in electronic structure, and τ₃ represents the partial recovery of the PChlide electronic ground state. Furthermore, τ₁ corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state.

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“…Light excitation of the enzyme-substrate complex induces the transfer of a hydride from the proS face of a NADPH molecule to the C-17 position of Pchlide, followed by proton donation to the C18 position by a tyrosine residue, finally leading to chlorophillide (Chlide), Scheme 12. 25,26 This reaction is an important step in the biosynthesis of chlorophyll.…”

Section: Lessons From Naturementioning

confidence: 99%

Catalytic processes activated by light

Galian

Pérez‐Prieto

2010

Energy Environ. Sci.

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Ultrafast catalytic processes and conformational changes in the light-driven enzyme protochlorophyllide oxidoreductase (POR)

Cited by 19 publications

References 41 publications

A Tale of Two Reductases: Extending the Bacteriochlorophyll Biosynthetic Pathway in E. coli

A Tale of Two Reductases: Extending the Bacteriochlorophyll Biosynthetic Pathway in E. coli

Excited-State Dynamics of Protochlorophyllide Revealed by Subpicosecond Infrared Spectroscopy

Catalytic processes activated by light

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