Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Mieke M E Huijbers,Marta Martínez-Júlvez,Milagros Medina,Willem J H Van Berkel,Estela Delgado-Arciniega,Adrie H Westphal

doi:10.1038/srep43880

Mieke M E Huijbers, Marta Martínez-Júlvez + Show 4 more

Open Access

https://doi.org/10.1038/srep43880

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Abstract

Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered.

Highlights

Flavoenzymes are ubiquitous in nature and function as versatile biocatalysts
We describe for the first time the production of fully reconstitutable apoprotein of proline dehydrogenase
Spectral analysis confirmed the presence of some holoenzyme, analogous to observations made with sarcosine oxidase and vanillyl-alcohol oxidase[28,29]

Summary

Introduction

Flavoenzymes are ubiquitous in nature and function as versatile biocatalysts They play an essential role in various biological processes such as biosynthesis, energy production, light emission, biodegradation, chromatin remodelling, DNA repair, apoptosis, protein folding, detoxification, and neural development[1]. Flavoenzymes usually contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as redox active prosthetic group (Fig. 1). These cofactors are synthesised from riboflavin (vitamin B2) through the action of riboflavin kinase (E.C. 2.7.1.26) and FMN adenylyltransferase (E.C. 2.7.7.2), respectively. While FMN enzymes show a preference for a TIM-barrel or flavodoxin-like fold, FAD enzymes often use a Rossmann fold for binding the ADP dinucleotide moiety of the cofactor[7]. Different methods have been explored to dissociate flavoproteins into apoprotein and flavin prosthetic group[16]. For flavoproteins with a more complex quaternary structure, production of fully reconstitutable apoprotein remains challenging

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