Essential enzymes in redox reactions, flavin reductases take part in a variety of cellular functions such as electron transport, biosynthesis, and detoxification. In this work, we conducted a comprehensive in silico characterization of a novel flavin reductase-like protein, REF, to examine its ligand-binding characteristics, structural organization, and evolutionary conservation. According to sequence-based domain analysis, REF is part of the flavin reductase-like domain and the FMN-binding split-barrel superfamily, indicating catalytic activity that is dependent on FMN. With conserved secondary structural elements and an RMSD in line with functional conservation, tertiary structure prediction showed that REF and a reference flavin reductase shared a high degree of structural similarity. Comparable molecular weights, isoelectric points, and hydropathicity were found by physiochemical analysis; however, REF showed a lower instability index, indicating greater structural stability. Strong binding affinity for both proteins was found by molecular docking of flavin mononucleotide (FMN), with REF showing a marginally higher predicted affinity (-9.1 kcal/mol) than flavin reductase (-8.8 kcal/mol). According to these results, REF shows modifications that could improve FMN binding while maintaining the structural integrity of flavin reductases. This study lays the groundwork for further experimental validation while highlighting the evolutionary and functional implications of such adaptations.

• Flavin Reductase; Fmn-Binding Protein; Evolutionary Conservation; Molecular Docking; Protein Modeling; Domain Analysis

Ali M (2025) Evolutionary Conservation and Structural Adaptations of Flavin Reductase-like Proteins: A Case Study on REF. J Bioinform, Genomics, Proteomics 7(1): 1043.

A functionally diverse group of enzymes known as flavin reductases are involved in electron transfer processes that support vital biological processes such as the reduction of toxic compounds, biosynthetic pathways, and the management of oxidative stress. Their use of flavin mononucleotide (FMN) as a cofactor and conserved structural fold make them members of the FMN-binding split-barrel superfamily. While adaptive modifications in primary structure can result in improved stability or changed substrate affinity, evolutionary conservation of this fold has allowed the retention of catalytic mechanisms across a variety of taxa [1]. The structural determinants of cofactor binding and activity can be better understood by comparing flavin reductases in silico. Variants with unique physiochemical characteristics are still less studied than the canonical flavin reductases, which are well-documented. In this study, we present a comparative bioinformatics-based analysis of REF, a recently discovered flavin reductase like protein, with an emphasis on its domain organization, evolutionary conservation, and structural adaptations that affect FMN binding [2].

Domain Analysis and Sequence Retrieval

The InterPro online portal (https://www.ebi.ac.uk/ interpro/) was used to analyze the amino acid sequence of REF by combining predictions from several domain and motif databases. Classifications by family and domain were contrasted with those of a known flavin reductase sequence.

Characterization by Physiochemistry

The ExPASy ProtParam tool (https://web.expasy. org/protparam/) was used to calculate physiochemical parameters, such as molecular weight (MW), theoretical isoelectric point (pI), grand average of hydropathicity (GRAVY), aliphatic index, and instability index.

Superimposition and Structure Prediction

Homology modeling techniques were used to predict the tertiary structures of flavin reductase and REF. Sites of mutation were indicated. To visualize structural conservation and evaluate root-mean-square deviation (RMSD), structural superimposition was used.

Docking of molecules

FMN docking simulations to both proteins were performed using AutoDock Vina. Potential variations in cofactor affinity were evaluated by analyzing predicted binding energies and binding site residues.

of Domains

According to InterPro analysis, flavin reductase and REF belong to the FMN-binding split-barrel superfamily, which also includes the flavin reductase-like domain. This arrangement implies functional conservation and is in line with FMN-dependent catalytic activity.

Properties

Characteristics              REF        Flavin reductase

No. of Amino Acids        205         205

Molecular Weight (Da)                 22581.222625.32

Theoretical pI                6.51        6.98

REF exhibited a slightly lower instability index, suggesting increased stability, with comparable hydrophobicity and aliphatic index to flavin reductase.

Comparing Structures

When tertiary structures were superimposed, the active-site architecture and secondary structure elements were conserved, and there was little variation in the backbone conformations. The effect of mutation sites on binding geometry was minimized because they were situated far from the FMN-binding pocket.

Docking of molecules

Strong FMN-binding affinities for both proteins were predicted by docking simulations:

Reductase of flavone: -8.8 kcal/mol Reference: -9.1 kcal/mol

Hydrophobic and hydrogen bonding interactions with conserved residues in the FMN-binding pocket were examples of binding interactions. Increased catalytic potential could be indicated by the marginally higher binding affinity for REF.

Our results show that conserved FMN-binding domains and tertiary structural organization, two characteristics of flavin reductases, are still present in REF. Together with conserved active-site residues, the minimal structural deviations point to strong evolutionary conservation. Subtle variations, like a slightly higher FMN-binding affinity and a lower instability index, could be adaptive changes that improve stability and cofactor interaction [3,4]. These adaptations might have developed as a result of selective pressures favoring more stable or effective redox-active enzymes, which could have been connected to metabolic or environmental factors specific to the organism that provided REF. REF might be a good option for applications needing strong flavin reductase activity because of the significance of FMN-dependent enzymes in industrial biocatalysis and detoxification [5,6].

With minor but potentially beneficial modifications, REF is a structurally conserved protein that resembles flavin reductase. REF is positioned as an intriguing target for additional biochemical and industrial application research due to its conserved catalytic architecture, improved stability, and marginally increased FMN-binding affinity.

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