The bacterial strains used in this study, Acinetobacter radioresistens M2Cr10 and Bacillus tropicus M54Cr10, were obtained from our laboratory culture collection. These strains were previously isolated and characterized by our group from samples of serpentine soil collected in South Kalimantan, Indonesia (Aisyah et al., 2017). They were selected for this study based on their demonstrated tolerance to high concentrations of chromium. All bacterial culturing was performed in Luria-Bertani (LB) Broth. Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, USA). Primers used to amplify the putative chromate reductase (chrR) genes are listed in Table 1. PCR amplifications were carried out using MyTaq™ HS Red Mix (Bioline, UK). Electrophoresis reagents included agarose, GelRed® (Biotium, USA), and a 100-base-pair (bp) DNA ladder (Vivantis, Malaysia). Bidirectional Sanger sequencing was performed by 1st BASE, Malaysia.

BLASTp analysis of the translated amino acid sequences, followed by phylogenetic analysis, classified both enzymes as members of the FMN-dependent nitroreductase superfamily Figure. 3. M2Cr10 was identified as an NADH-ubiquinone oxidoreductase (NfsB-like family), while M54Cr10 was classified as an NADPH-dependent FMN reductase (NfsA-like family). Genetic divergence analysis Figure. 4 indicated that M2Cr10 and M54Cr10 do not form a distinct clade but are interspersed with known nitroreductases from various bacteria, including Bacillus subtilis and Escherichia coli. These findings support the hypothesis that the enzymes are not specialized chromate reductases but rather multifunctional nitroreductases capable of adventitious Cr(VI) reduction.

Bootstrap-supported clustering (1,000 replicates) positioned M2Cr10 within Clade I alongside Bacillus subtilis NfrA1 (PDB: 3N2S), Escherichia coli K12 (PDB: 1F5V), and Vibrio harveyi (PDB: 1BKJ). M54Cr10 clustered closely with Bacillus megaterium NfrA1 (PDB: 5HDJ) and Bacillus subtilis (PDB: 1ZCH), exhibiting genetic divergence distances of 0.816 - 1.086. These distances, calculated using the Poisson correction model, reflect conserved functional domains critical for chromate reduction.

The structural homology of M2Cr10 and M54Cr10 with nitroreductase-like proteins (e.g., Bacteroides fragilis NCTC 9343 [PDB: 3EOF], Parabacteroides distasonis ATCC 8503 [PDB: 3M5K]) suggests shared catalytic mechanisms, particularly in FMN-dependent redox reactions. Notably, M54Cr10’s alignment with Priestia megaterium NfrA2 (PDB: 5HEI) highlights the evolutionary conservation of NADPH-binding motifs, whereas M2Cr10’s proximity to Geobacter metallireducens GS-15 (PDB: 4DN2) indicates adaptive divergence in electron transport pathways.

The predicted physicochemical properties of the two enzymes suggest distinct structural characteristics Table 5. M2Cr10 has a predicted molecular weight of 26.56 kDa and a theoretical pI of 5.89, while M54Cr10 is larger (28.72 kDa) with a higher pI (6.27). The instability index, which predicts in vivo stability, was 36.87 for M2Cr10 and 40.82 for M54Cr10. Values below 40 indicate stable proteins, while values above 40 suggest potential instability, indicating M2Cr10 is likely stable, whereas M54Cr10 may be less stable (Guruprasad et al., 1990). Both enzymes exhibited negative Grand Average of Hydropathy (GRAVY) scores, consistent with hydrophilic proteins and supporting their cytosolic localization and function in an aqueous environment.

High-quality 3D homology models were generated for both enzymes Figure. 5. Validation using PROCHECK confirmed excellent stereochemical quality. The M2Cr10 model contained 96.1% of residues in the most favored regions of the Ramachandran plot, whereas the M54Cr10 model contained 95.8%, with no residues in disallowed regions for either model. These results indicated that both models are reliable and provide a solid foundation for subsequent docking and molecular dynamics analyses (Martin et al., 2015).

The structural conservation of these active-site residues underscores evolutionary optimization for chromate reduction, with α/β folds stabilizing cofactor binding and catalytic loops. Further site-directed mutagenesis studies are warranted to validate the functional roles of these residues in Cr(VI) detoxification (Carles et al. 2016).

Molecular docking of FMN with chromate reductases from Acinetobacter radioresistens M2Cr10 and Bacillus tropicus M54Cr10 identified distinct binding residues and energy profiles Table 6; Figure 6. In M2Cr10, FMN interactions involved His¹¹, Ser¹³, Arg¹5, Ser39, Ser40, His41, Ala38, Asn66, Tyr¹³¹, Gly¹³³, and Lys170, resulting in a binding energy of -7.1 kcal/mol. In contrast, M54Cr10 exhibited interactions at Arg15, Ser39, Ser40, Phe42, Asn66, Tyr¹³¹, Gly¹³³, and Gly134, with a lower binding energy of -8.1 kcal/mol for M54Cr10, indicating stronger ligand affinity.

Hydrogen bonding and hydrophobic interactions dominated the binding mechanisms. In M2Cr10, Arg15 formed a hydrogen bond with the FMN ligand (3.25 Å), while Tyr¹³¹ and Lys170 engaged in hydrophobic contacts. In M54Cr10, Ser39 formed a hydrogen bond (3.26 Å), and Phe42 contributed to hydrophobic stabilization (4.96 Å). The greater residue diversity within M2Cr10’s binding pocket suggests broader interaction networks, whereas the more compact binding site in M54Cr10’s may enhance ligand-binding efficiency.

Binding energy, a critical determinant of ligand-protein stability, reflects the thermodynamic favorability of interactions (Putra et al., 2023); Jakhmola et al. 2022). Molecular docking predicted that FMN binds favorably within the active site of both enzymes, with binding energies of -7.1 kcal/ mol for M2Cr10 and -8.1 kcal/mol for M54Cr10, indicating stronger affinity in M54Cr10. In both enzymes, the FMN cofactor is stabilized by a network of hydrogen bonds and hydrophobic interactions. Energy decomposition analysis revealed a key role for residue Tyr¹³¹, which provides significant hydrophobic and π-stacking interactions with the isoalloxazine ring of FMN, acting as a crucial anchor. This observation highlights a conserved structural feature for cofactor stabilization within this enzyme family.

Energy decomposition analysis using the FastRDH web server Figure 7 identified Tyr¹³¹ as a critical residue governing flavin mononucleotide (FMN) binding in both Acinetobacter radioresistens M2Cr10 and Bacillus tropicus M54Cr10. The exceptionally low interaction energy (−ΔG) associated with Tyr¹³¹ highlights its role in stabilizing FMN through hydrophobic interactions, a mechanism not previously reported for chromate reductases. Hydrophobic exclusion of water molecules by Tyr¹³¹ significantly enhanced binding stability, as evidenced by energy profiles and molecular dynamics trajectories.

This novel finding highlights Tyr¹³¹ as a conserved anchor within FMN-binding pockets, providing insights into evolutionary optimization of redox cofactor retention. The lack of prior reports on Tyr¹³¹’s contribution to FMN affinity highlights the uniqueness of these enzymes’ ligand-binding architecture. These results provide a structural framework for the rational engineering of chromate reductases, enabling targeted modifications to augment cofactor binding efficiency for enhanced Cr(VI) reduction.

To compare the structural dynamics, 10 ns molecular dynamics (MD) simulations were performed. The RMSD of the protein backbone atoms was monitored to assess overall structural stability Figure 8. The M2Cr10 enzyme rapidly reached equilibrium and maintained a lower and more stable RMSD (average ~0.45 nm) throughout the simulation, whereas M54Cr10 showed greater deviation and conformational drift (average ~0.60 nm). The results indicate that M2Cr10 has a more rigid and stable global structure.

Initial conformational divergence during the first 0 - 3 ns of the simulation suggested distinct structural sampling pathways between the two enzymes. From 3 - 10 ns, M2Cr10 exhibited consistently lower RMSD values (mean: 1.2 ± 0.3 Å) compared to M54Cr10 (mean: 2.8 ± 0.6 Å), indicating enhanced structural rigidity and reduced conformational flexibility in M2Cr10.The pronounced RMSD fluctuations in M54Cr10 correlate with its higher aliphatic index (74.78 vs. 77.09 for M2Cr10), suggesting that reduced thermostability may permit greater loop mobility. These observations align with stability indices predicted by ProtParam (M2Cr10: 36.87; M54Cr10: 40.82), where lower indices denote higher stability (Cunha et al., 2015). The structural rigidity of M2Cr10 implies evolutionary optimization for catalytic consistency, whereas the flexibility of M54Cr10 may facilitate adaptive substrate binding.

Root mean square fluctuation (RMSF) analysis was performed to evaluate residue-specific flexibility in Acinetobacter radioresistens M2Cr10 and Bacillus tropicus M54Cr10 during 10 ns molecular dynamics simulations Figure 9. M2Cr10 exhibited higher RMSF values (mean: 1.8 ± 0.4 Å) when compared with M54Cr10 (mean: 1.2 ± 0.3 Å), particularly in residues 170 – 200 and 225 – 250, indicating enhanced flexibility in loop regions critical for substrate binding. In contrast, M54Cr10 displayed reduced fluctuations, suggesting structural rigidity that may contribute to the stabilization of FMN cofactor interactions.

This observation was further supported by the Solvent Accessible Surface Area (SASA) analysis, in which M2Cr10 consistently showed lower values, indicating a more compact fold (Fig. 10C). The Radius of Gyration (Rg) was comparable for both enzymes, although M54Cr10 displayed a slightly more compact core Figure. 10A & B.

Analysis of local flexibility via RMSF revealed that, despite its global stability, M2Cr10 exhibits significantly higher flexibility in specific loop regions (residues ~170–200)comparedtothemorerigidM54Cr10. These flexible loops, located near the active site, are critical for substrate access and product release. These findings suggest a functional trade-off:M2Cr10 combine sastable structural scaffold with dynamic, flexible loops, an architecture often associated with efficient catalysis in robust enzymes.