Structure, mechanism, and evolution of the last step in vitamin C biosynthesis.

Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function.

Evolution of the catalytic mechanism at the dawn of the Baeyer-Villiger monooxygenases.

Enzymes are crucial for the emergence and sustenance of life on earth. How they became catalytically active during their evolution is still an open question. Two opposite explanations are plausible: acquiring a mechanism in a series of discrete steps or all at once in a single evolutionary event. Here, we use molecular phylogeny, ancestral sequence reconstruction, and biochemical characterization to follow the evolution of a specialized group of flavoprotein monooxygenases, the bacterial Baeyer-Villiger monooxygenases (BVMOs). These enzymes catalyze an intricate chemical reaction relying on three different elements: a reduced nicotinamide cofactor, dioxygen, and a substrate. Characterization of ancestral BVMOs shows that the catalytic mechanism evolved in a series of steps starting from a FAD-binding protein and further acquiring reactivity and specificity toward each of the elements participating in the reaction. Together, the results of our work portray how an intrinsically complex catalytic mechanism emerged during evolution.

In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis.

Metabolons are protein assemblies that perform a series of reactions in a metabolic pathway. However, the general importance and aptitude of metabolons for enzyme catalysis remain poorly understood. In animals, biosynthesis of coenzyme Q is currently attributed to ten different proteins, with COQ3, COQ4, COQ5, COQ6, COQ7 and COQ9 forming the iconic COQ metabolon. Yet several reaction steps conducted by the metabolon remain enigmatic. To elucidate the prerequisites for animal coenzyme Q biosynthesis, we sought to construct the entire metabolon in vitro. Here we show that this approach, rooted in ancestral sequence reconstruction, reveals the enzymes responsible for the uncharacterized steps and captures the biosynthetic pathway in vitro. We demonstrate that COQ8, a kinase, increases and streamlines coenzyme Q production. Our findings provide crucial insight into how biocatalytic efficiency is regulated and enhanced by these biosynthetic engines in the context of the cell.

Impact of ancestral sequence reconstruction on mechanistic and structural enzymology.

Ancestral sequence reconstruction (ASR) provides insight into the changes within a protein sequence across evolution. More specifically, it can illustrate how specific amino acid changes give rise to different phenotypes within a protein family. Over the last few decades it has established itself as a powerful technique for revealing molecular common denominators that govern enzyme function. Here, we describe the strength of ASR in unveiling catalytic mechanisms and emerging phenotypes for a range of different proteins, also highlighting biotechnological applications the methodology can provide.

Identification and characterization of archaeal and bacterial F420 -dependent thioredoxin reductases.

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F420 H2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed that these two DFTRs are highly specific for F420 H2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR.

Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives.

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for É£-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.

Evolution of enzyme functionality in the flavin-containing monooxygenases.

Among the molecular mechanisms of adaptation in biology, enzyme functional diversification is indispensable. By allowing organisms to expand their catalytic repertoires and adopt fundamentally different chemistries, animals can harness or eliminate new-found substances and xenobiotics that they are exposed to in new environments. Here, we explore the flavin-containing monooxygenases (FMOs) that are essential for xenobiotic detoxification. Employing a paleobiochemistry approach in combination with enzymology techniques we disclose the set of historical substitutions responsible for the family's functional diversification in tetrapods. Remarkably, a few amino acid replacements differentiate an ancestral multi-tasking FMO into a more specialized monooxygenase by modulating the oxygenating flavin intermediate. Our findings substantiate an ongoing premise that enzymatic function hinges on a subset of residues that is not limited to the active site core.

Evolutionary and structural analyses of the NADPH oxidase family in eukaryotes reveal an initial calcium dependency.

Reactive oxygen species are unstable molecules generated by the partial reduction of dioxygen. NADPH oxidases are a ubiquitous family of enzymes devoted to ROS production. They fuel an array of physiological roles in different species and are chemically demanding enzymes requiring FAD, NADPH and heme prosthetic groups in addition to either calcium or a various number of cytosolic mediators for activity. These activating partners are exclusive components that partition and distinguish the NOX members from one another. To gain insight into the evolution of these activating mechanisms, and in general in their evolutionary history, we conducted an in-depth phylogenetic analysis of the NADPH oxidase family in eukaryotes. We show that all characterized NOXs share a common ancestor, which comprised a fully formed catalytic unit. Regarding the activation mode, we identified calcium-dependency as the earliest form of NOX regulation. The protein-protein mode of regulation would have evolved more recently by gene-duplication with the concomitant loss of the EF-hands motif region. These more recent events generated the diversely activated NOX systems as observed in extant animals and fungi.

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency.

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

Resurrecting Enzymes by Ancestral Sequence Reconstruction.

Ancestral Sequence Reconstruction (ASR) allows one to infer the sequences of extinct proteins using the phylogeny of extant proteins. It consists of disclosing the evolutionary history-i.e., the phylogeny-of a protein family of interest and then inferring the sequences of its ancestors-i.e., the nodes in the phylogeny. Assisted by gene synthesis, the selected ancestors can be resurrected in the lab and experimentally characterized. The crucial step to succeed with ASR is starting from a reliable phylogeny. At the same time, it is of the utmost importance to have a clear idea on the evolutionary history of the family under study and the events that influenced it. This allows us to implement ASR with well-defined hypotheses and to apply the appropriate experimental methods. In the last years, ASR has become popular to test hypotheses about the origin of functionalities, changes in activities, understanding physicochemical properties of proteins, among others. In this context, the aim of this chapter is to present the ASR approach applied to the reconstruction of enzymes-i.e., proteins with catalytic roles. The spirit of this contribution is to provide a basic, hands-to-work guide for biochemists and biologists who are unfamiliar with molecular phylogenetics.

On the diversity of F420 -dependent oxidoreductases: A sequence- and structure-based classification.

The F420 deazaflavin cofactor is an intriguing molecule as it structurally resembles the canonical flavin cofactor, although behaves as a nicotinamide cofactor due to its obligate hydride-transfer reactivity and similar low redox potential. Since its discovery, numerous enzymes relying on it have been described. The known deazaflavoproteins are taxonomically restricted to Archaea and Bacteria. The biochemistry of the deazaflavoenzymes is diverse and they exhibit great structural variability. In this study a thorough sequence and structural homology evolutionary analysis was performed in order to generate an overarching classification of the F420 -dependent oxidoreductases. Five different deazaflavoenzyme Classes (I-V) are described according to their structural folds as follows: Class I encompassing the TIM-barrel F420 -dependent enzymes; Class II including the Rossmann fold F420 -dependent enzymes; Class III comprising the ß-roll F420 -dependent enzymes; Class IV which exclusively gathers the SH3 barrel F420 -dependent enzymes and Class V including the three layer ßßα sandwich F420 -dependent enzymes. This classification provides a framework for the identification and biochemical characterization of novel deazaflavoenzymes.

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties.

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

Publisher Correction: Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Broadening the repertoire of microbial aldo-keto reductases: cloning and characterization of AKR3B4 from Rhodotorula mucilaginosa LSL strain.

Aldo-keto reductases (AKRs) are nicotinamide-dependent enzymes that catalyze the transformation of aldehydes and ketones into alcohols. They are spread across all phyla, and those from microbial origin have proved to be highly robust and versatile biocatalysts. In this work, we have discovered and characterized a microbial AKR from the yeast Rhodotorula mucilaginosa by combining genome-mining and expression assays. The new enzyme, named AKR3B4, was expressed by a simple protocol in very good amounts. It displays a selective substrate profile exclusively transforming aldehydes into alcohols. Also, AKR3B4 shows very good stability at medium temperatures, in a broad range of pH values and in the presence of green organic solvents. Conversion assays demonstrate it is an excellent biocatalyst to be used in the synthesis of aromatic alcohols, and also to produce furan-3-ylmethanol and the valuable sweetener xylitol. These results show that AKR3B4 displays attractive features so as to be used in chemoenzymatic processes.

Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs.

Flavin-containing monooxygenases (FMOs) are ubiquitous in all domains of life and metabolize a myriad of xenobiotics, including toxins, pesticides and drugs. However, despite their pharmacological importance, structural information remains bereft. To further our understanding behind their biochemistry and diversity, we used ancestral-sequence reconstruction, kinetic and crystallographic techniques to scrutinize three ancient mammalian FMOs: AncFMO2, AncFMO3-6 and AncFMO5. Remarkably, all AncFMOs could be crystallized and were structurally resolved between 2.7- and 3.2-Å resolution. These crystal structures depict the unprecedented topology of mammalian FMOs. Each employs extensive membrane-binding features and intricate substrate-profiling tunnel networks through a conspicuous membrane-adhering insertion. Furthermore, a glutamate-histidine switch is speculated to induce the distinctive Baeyer-Villiger oxidation activity of FMO5. The AncFMOs exhibited catalysis akin to human FMOs and, with sequence identities between 82% and 92%, represent excellent models. Our study demonstrates the power of ancestral-sequence reconstruction as a strategy for the crystallization of proteins.

Plant tissue cultures as sources of new ene- and ketoreductase activities.

While many redox enzymes are nowadays available for synthetic applications, the toolbox of ene-reductases is still limited. Consequently, the screening for these enzymes from diverse sources in the search of new biocatalyst suitable for green chemistry approaches is needed. Among 13 plant tissue cultures, Medicago sativa and Tessaria absinthioides calli, as well as Capsicum annuum hairy roots, were selected due to their ability to hydrogenate the CC double bond of the model substrate 2-cyclohexene-1-one. The three axenic plant cultures showed more preference toward highly activated molecules such as nitrostyrene and maleimide rather than the classical substrates of the well-known Old Yellow Enzymes, resembling the skills of the NAD(P)H-dependent flavin-independent enzymes. When the three biocatalytic systems were applied in the reduction of chalcones, T. absinthioides showed high chemoselectivity toward the CC double bond whereas the other two demonstrated abilities to biohydrogenate the CC double bounds and the carbonyl groups in a sequential fashion.

Chopping and Changing: the Evolution of the Flavin-dependent Monooxygenases.

Flavin-dependent monooxygenases play a variety of key physiological roles and are also very powerful biotechnological tools. These enzymes have been classified into eight different classes (A-H) based on their sequences and biochemical features. By combining structural and sequence analysis, and phylogenetic inference, we have explored the evolutionary history of classes A, B, E, F, and G and demonstrate that their multidomain architectures reflect their phylogenetic relationships, suggesting that the main evolutionary steps in their divergence are likely to have arisen from the recruitment of different domains. Additionally, the functional divergence within in each class appears to have been the result of other mechanisms such as a complex set of single-point mutations. Our results reinforce the idea that a main constraint on the evolution of cofactor-dependent enzymes is the functional binding of the cofactor. Additionally, a remarkable feature of this family is that the sequence of the key flavin adenine dinucleotide-binding domain is split into at least two parts in all classes studied here. We propose a complex set of evolutionary events that gave rise to the origin of the different classes within this family.

The Origin and Evolution of Baeyer-Villiger Monooxygenases (BVMOs): An Ancestral Family of Flavin Monooxygenases.

The Baeyer-Villiger Monooxygenases (BVMOs) are enzymes belonging to the "Class B" of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga) and Haptophyta (Emiliania huxleyi) for the first time. Furthermore, a search for other "Class B" monooxygenases (flavoprotein monooxygenases--FMOs--and N-hydroxylating monooxygenases--NMOs) was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all "Class B" monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA) and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes.

Insights in the kinetic mechanism of the eukaryotic Baeyer-Villiger monooxygenase BVMOAf1 from Aspergillus fumigatus Af293.

This work reports a detailed kinetic study of the recently discovered BVMOAf1 from Aspergillus fumigatus Af293. By performing steady state and pre-steady state kinetic analyses, it was demonstrated that the rate of catalysis is partially limited by the NADPH-mediated reduction of the flavin cofactor, a unique hallmark of BVMOAf1. In addition, the oxygenating C4a-(hydro)peroxyflavin intermediate could be spectrophotometrically detected and it was found to be the most stable among all analyzed BVMOs. To assess the possible influence of some residues on the kinetic features, model-inspired site-directed mutagenesis was performed. Among the mutants, the Q436A variant showed a slightly broader substrate scope and a better catalytic efficiency. In summary, this study describes for the first time the kinetic parameters for an eukaryotic BVMO.