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Puromycin N AcetyltransferaseEdit

Puromycin N Acetyltransferase is a small, cytosolic enzyme that inactivates the antibiotic puromycin by acetylating its amino group, thereby granting cells carrying the encoding gene resistance to puromycin. The enzyme is encoded by the pac gene and is widely used in molecular biology as a selectable marker in genetic engineering, including in mammalian cell culture and bacterial systems. Its activity places Puromycin N Acetyltransferase squarely within the family of acetyltransferases, specifically the GCN5-related N-acetyltransferase (GNAT) superfamily, which uses acetyl-CoA as a donor to modify substrates through acetylation. For more on the substrate and family, see Puromycin and GNAT.

Puromycin, an antibiotic that mimics aminoacyl-tRNA, disrupts protein synthesis by causing premature chain termination. Puromycin N acetyltransferase counters this effect by converting puromycin into N-acetylpuromycin, a form that no longer interferes with ribosome function. This mechanism makes pac-bearing cells capable of surviving puromycin exposure under controlled laboratory conditions. The relationship between the drug and the resistance enzyme is a classic example of how a selectable marker functions in genetic engineering and cell biology workflows; see Selectable marker for broader context.

Biochemical properties and mechanism

  • Substrate and product: The enzyme acts on puromycin to produce N-acetylpuromycin, inactivating the antibiotic’s ability to disrupt translation. See Puromycin for background on the drug’s mode of action.
  • Cofactor: The acetyl donor is acetyl-CoA, as is typical for GNAT family members. For a broader view of this class, consult GNAT.
  • Enzymatic class: Puromycin N acetyltransferase is considered a small, monomeric cytosolic enzyme with a preference for puromycin, though related GNAT enzymes exhibit broad substrate scopes in other contexts. More on acetyltransferases can be found in Acetyltransferase.

Genetics, origin, and distribution

  • Gene and encoding context: pac is the gene that encodes Puromycin N Acetyltransferase. In laboratory plasmids and vectors, pac serves as a reliable selectable marker to identify cells that have incorporated a construct of interest. See Plasmid and Selectable marker for related concepts.
  • Distribution and use: Pac is exported in commonly used research vectors across prokaryotic and eukaryotic systems, including mammalian cell lines and, in some cases, yeast or other model organisms. See Mammalian cell and Cell culture for related topics.
  • Evolutionary note: The pac gene is derived from bacterial sources, consistent with many antibiotic resistance determinants used in biotechnology. The ongoing use of pac reflects a long-standing practice in genetics research: a practical tool with well-established protocols and safety practices.

Structure and properties (overview)

  • Token size and fold: Puromycin N Acetyltransferase is a relatively small enzyme, typical of GNAT family members, with a catalytic core designed to accommodate acetyl-CoA and puromycin in proximity to enable acetyl transfer. For structural context, see GNAT.
  • Catalytic features: The active site coordinates the acetyl donor and substrate to effect the transfer, producing N-acetylpuromycin. Readers interested in structural studies of similar enzymes can consult articles on GNAT family members in GNAT.

Applications in biotechnology

  • Selectable marker: In genetic engineering, pac allows researchers to select for cells that have integrated a vector of interest by providing resistance to puromycin. This is a standard alternative to antibiotic resistance markers used in various systems, and it is widely adopted in lab protocols. See Selectable marker for broader usage.
  • Mammalian cell culture workflows: Puromycin selection is valued for its rapid action and relative simplicity in many mammalian cell systems, contributing to efficient isolation of transfected or edited cells. See Mammalian cell and Cell culture for related context.
  • Safety and regulatory considerations: The use of antibiotic resistance markers in laboratory research is subject to biosafety oversight and institutional policies. The pac system is generally restricted to controlled environments, with regulatory guidance guiding its use in experiments and product development. See Biosafety and Biosecurity for broader discussions.

Controversies and debates (from the practical, policy-oriented perspective)

  • Antibiotic resistance concerns: Critics worry that ever-present antibiotic resistance genes in the lab could contribute to broader resistance issues if misused or released. Proponents counter that in controlled laboratory settings with proper containment, institutional oversight, and non-environmental release controls, the risk is managed and the benefits in enabling precise genetic work justify the approach. See Antibiotic resistance for general background.
  • Alternatives and trade-offs: Some researchers advocate for non-antibiotic selectable markers (such as fluorescent reporters or metabolic complementation systems) to reduce reliance on antibiotic resistance markers. Others point out that antibiotic-based markers like pac offer robust, fast, and scalable selection in many contexts, particularly in complex mammalian cell work. See Selectable marker and Genetic engineering for related discussions.
  • Policy and public discourse: In broader public debates, efficiency, cost, and scientific progress often compete with precautionary concerns. Advocates of streamlined research argue that well-regulated lab practices, transparent risk assessment, and credible safety data reduce real-world risk, while critics may label any use of antibiotic resistance markers as unnecessary risk. The practical stance emphasizes disciplined oversight, risk-based evaluation, and ongoing appraisal of alternative methods.

See also