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Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

ABSTRACT: The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.

SUBMITTER: Rodriguez Buitrago JA

PROVIDER: S-EPMC7397466 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

Rodriguez Buitrago Jhon Alexander JA Klünemann Thomas T Blankenfeldt Wulf W Schallmey Anett A

Acta crystallographica. Section F, Structural biology communications 20200728 Pt 8

The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolutio ...[more]

PMID: 32744244

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Project description:BackgroundCellulose, an abundant biopolymer, has great potential to be utilized as a renewable fuel feedstock through its enzymatic degradation into soluble sugars followed by sugar fermentation into liquid biofuels. However, crystalline cellulose is highly resistant to hydrolysis, thus industrial-scale production of cellulosic biofuels has been cost-prohibitive to date. Mechanistic studies of enzymes that break down cellulose, called cellulases, are necessary to improve and adapt such biocatalysts for implementation in biofuel production processes. Thermobifida fusca Cel6B (TfCel6B) is a promising candidate for industrial use due to its thermostability and insensitivity to pH changes. However, mechanistic studies probing TfCel6B hydrolytic activity have been limited to ensemble-scale measurements.ResultsWe utilized optical tweezers to perform single-molecule, nanometer-scale measurements of enzyme displacement during cellulose hydrolysis by TfCel6B. Records featured forward motility on the order of 0.17 nm s-1 interrupted by backward motions and long pauses. Processive run lengths were on the order of 5 nm in both forward and backward directions. Motility records also showed rapid bidirectional displacements greater than 5 nm. Single-enzyme velocity and bulk ensemble activity were assayed on multiple crystalline cellulose allomorphs revealing that the degree of crystallinity and hydrogen bonding have disparate effects on the single-molecule level compared to the bulk scale. Additionally, we isolated and monitored the catalytic domain of TfCel6B and observed a reduction in velocity compared to the full-length enzyme that includes the carbohydrate-binding module. Applied force has little impact on enzyme velocity yet it readily facilitates dissociation from cellulose. Preliminary measurements at elevated temperatures indicated enzyme velocity strongly increases with temperature.ConclusionsThe unexpected motility patterns of TfCel6B are likely due to previously unknown mechanisms of processive cellulase motility implicating irregularities in cellulose substrate ultrastructure. While TfCel6B is processive, it has low motility at room temperature. Factors that most dramatically impact enzyme velocity are temperature and the presence of its native carbohydrate-binding module and linker. In contrast, substrate ultrastructure and applied force did not greatly impact velocity. These findings motivate further study of TfCel6B for its engineering and potential implementation in industrial processes.

Dataset Information

Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

Publications

Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

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