Recombinant Rhinolophus ferrumequinum Histone-lysine N-methyltransferase setd3 (SETD3), partial

Molecular Identity and Conservation

SETD3 belongs to the SET-domain methyltransferase family, initially misclassified as a histone-lysine methyltransferase but later identified as the first actin-specific histidine N-methyltransferase in humans, rats, and Drosophila . Key features include:

• Catalytic domain: A conserved SET domain responsible for methyl transfer from S-adenosyl methionine (SAM) to substrates .

• Substrate specificity: Preferentially methylates histidine residues (e.g., H73 in β-actin) over lysine, with a 10–4,000-fold higher activity for histidine-containing peptides .

• Structural conservation: Bat SETD3 likely shares >80% sequence homology with human SETD3, given evolutionary conservation among mammals .

Table 1: Conserved Features of SETD3 Across Species

Figure 1: Proposed Catalytic Mechanism (Inferred from Human SETD3)

1. Substrate docking: Actin’s residues 69–77 bind to SETD3’s surface.

2. Histidine positioning: H73 imidazole ring rotates to align Nτ with SAM’s methyl group.

3. Methyl transfer: Direct transfer occurs via an $S_N2$-like mechanism .

Enzymatic Activity and Kinetic Parameters

While bat SETD3 remains uncharacterized, human SETD3 kinetics provide a benchmark:

• Optimal conditions: pH 7.5–8.0, 25–37°C, dependent on Mg²⁺ .

• Substrate kinetics:

  $$K_m \, (\text{β-actin}) = 0.8 \, \mu\text{M}, \quad k_{cat} = 9.6 \, \text{min}^{-1} \, \text{[1]}$$

• Inhibition: Competitive inhibition by sinefungin (SAM analog) with $IC_{50} = 2.3 \, \mu\text{M}$ .

Biological and Pathological Relevance

SETD3-mediated actin methylation stabilizes F-actin filaments and regulates cytoskeletal dynamics. Loss of SETD3 in model systems leads to:

• Cellular effects: Reduced F-actin content (+15% glycolytic flux) and impaired cell motility .

• Physiological impacts: Maternal dystocia in mice due to uterine smooth muscle dysfunction .

Table 2: Phenotypic Consequences of SETD3 Knockout

Research Applications of Recombinant SETD3

The partial bat enzyme could be used for:

1. Comparative studies: Testing cross-species activity on actin isoforms.

2. Drug discovery: Screening for SETD3 inhibitors targeting cytoskeletal disorders.

3. Enzyme engineering: Creating lysine-specific variants for biotechnological applications .

Open Questions and Future Directions

1. Does bat SETD3 exhibit unique substrate preferences compared to mammalian orthologs?

2. What role does actin methylation play in bat-specific biology (e.g., echolocation musculature)?

3. Can bat SETD3’s partial structure inform minimal catalytic domain requirements?