Structural Adaptations of Bacterial Grx3 to Temperature: Pro29 Is Essential for Cold Adaptation in Sphingomonas sp. Grx3

Abstract

Bacterial glutaredoxin 3 (Grx3) is a class I oxidoreductase with a canonical thioredoxin (Trx) fold, yet its thermal adaptation mechanisms remain unclear. We investigated cold adaptation in the psychrophilic ortholog from the Arctic bacterium Sphingomonas sp. (SpGrx3). While mesophilic orthologs, such as Escherichia coli Grx3 (EcGrx3), typically feature an α1-β2 salt bridge (Lys19-Glu31), SpGrx3 lacks this bridge due to Leu19, although Glu31 is conserved. The L19K mutation in SpGrx3 failed to form the salt bridge, yielding the least stable mutant. However, the introduction of a P29F or P29Y substitution in the α1 loop in combination with L19K restored the salt bridge and significantly enhanced both the melting temperature and stability. Phe29 stabilizes the structure via hydrophobic interactions, enhancing substrate affinity, while Tyr29 enhances catalytic rates through hydrogen bonding. Conversely, the reciprocal F29P substitution in EcGrx3 disrupted the salt bridge and markedly reduced its melting temperature. Notably, K19L/F29P, which mimics the SpGrx3 wild-type (WT) configuration, increased melting temperature via Leu19 hydrophobic interactions, similar to F29Y with the salt bridge. These results underscore the crucial role of Phe or Tyr at position 29 in forming the Lys19-Glu31 salt bridge in warmer-temperature orthologs and suggest that the transition to Pro is a critical adaptation in psychrophilic orthologs. This study provides new insights into Grx3's structural adaptations to varying thermal habitats through diverse α1-β2 interactions.

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Sequence and structure comparison of Grx3 orthologs. (A) Multiple sequence alignment of Grx3 members from various temperature environments. The active-site CXXC motif is highlighted in an orange box. Residues at positions 19 and 31, forming the α1−β2 salt bridge in mesophilic and thermophilic species, are marked in green. Residues at positions 23 and 29, forming the intraloop hydrogen bond in some mesophilic and psychrophilic orthologs, are marked in blue. Psychrophilic Grx3s: SpGrx3 (Sphingomonas sp. PAMC 26621, NCBI ID: WP_010217562.1), ArGrx3 (Arthrobacter sp. TPD3018, NCBI ID: PVE52959.1), CpGrx3 (Colwellia piezophile, NCBI ID: WP_019027132.1); Mesophilic Grx3s: EcGrx3 (Escherichia coli K-12, PDB ID: 1FOV), DeGrx3 (Desulfuromonadales bacterium, NCBI ID: NIQ93024.1), AbGrx3 (Azospirillum brasilense, NCBI ID: WP_200477686.1); Thermophilic Grx3s: CoGrx3 (Chromatium okenii, NCBI ID: WP_105074337.1), RpGrx3 (Rhodopseudomonas palustris, NCBI ID: WP_110787285.1), TtGrx3 (Thermochromatium tepidum ATCC 43061, NCBI ID: QGU33019.1). (B) Structural model of SpGrx3. The two catalytic Cys residues are shown in yellow. Residues in the aliphatic cluster are depicted in green, and those in the aromatic cluster in orange. (C,D) Enlarged views of the α1−β2 regions of SpGrx3 and EcGrx3, respectively.

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Conformational stability of SpGrx3 WT and mutants. Fluorescence spectra were recorded after 30 min incubation at 25 °C with 0–8 M urea (excitation at 275 nm). Data represents the mean ± SD of three experiments.

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Acrylamide Stern–Volmer plot of SpGrx3 WT and mutants. Fluorescence was recorded as the maximum intensity in the absence (F ₀) and presence (F) of acrylamide, and the ratio F ₀/F was plotted. Data represents the mean ± SD of three experiments.

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Far-UV CD spectra of SpGrx3 WT and mutants at 25 °C.

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RMSD and RMSF analysis of SpGrx3 WT and mutants. (A) C _α RMSD over time. (B) C _α RMSF per residue, with secondary structure elements annotated.

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Far-UV CD spectra of EcGrx3 C66Y and its mutants at 25 °C.

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RMSD and RMSF analysis of EcGrx3 C66Y and its mutants. (A) C _α RMSD over time. (B) C _α RMSF per residue, with secondary structure elements annotated.

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Predicted pathways of Grx3 adaptation to colder temperatures. (A) The predicted ancestral Grx3 possessed both the Lys19-Glu31 salt bridge and the Lys23-Tyr29 hydrogen bond. Pathway 1: As Grx3 evolved to cooler climates, it first underwent a Y29F substitution (B), leaving Lys23 unpaired (as seen in thermophilic Grx3, e.g., CoGrx3). Later, mesophilic orthologs, such as StGrx3, acquired a K23A substitution (C), and psychrophilic orthologs eventually adopted an F29P substitution that disrupted the salt bridge, followed by a K19L change (E) (e.g., SpGrx3). Pathway 2: Some mesophilic Grx3s acquired a K23D substitution (D), forming an Asp23-Tyr29 hydrogen bond while retaining the salt bridge (e.g., DeGrx3). In this pathway, psychrophilic orthologs replaced the salt bridge with an Arg19-Asn31 hydrogen bond (F) (e.g., PhGrx3). Pathway 3: Certain psychrophilic orthologs diverged directly from the ancestral Grx3 without intermediate mesophilic forms. These either feature Leu19 with an intraloop Lys23-Tyr29 hydrogen bond (G) (e.g., CpGrx3) or retain both the salt bridge and Lys23-Tyr29 hydrogen bond (H) (e.g., PsGrx3).