June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Structural and Functional Adaptations in N1pC/P60 Enzymes Facilitate Retinyl Ester Formation
Author Affiliations & Notes
  • Marcin Golczak
    Pharmacology, Case Western Reserve University, Cleveland, OH
  • Avery E. Sears
    Pharmacology, Case Western Reserve University, Cleveland, OH
  • Philip D. Kiser
    Pharmacology, Case Western Reserve University, Cleveland, OH
  • Krzysztof Palczewski
    Pharmacology, Case Western Reserve University, Cleveland, OH
  • Footnotes
    Commercial Relationships Marcin Golczak, None; Avery Sears, None; Philip Kiser, None; Krzysztof Palczewski, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5517. doi:
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      Marcin Golczak, Avery E. Sears, Philip D. Kiser, Krzysztof Palczewski; Structural and Functional Adaptations in N1pC/P60 Enzymes Facilitate Retinyl Ester Formation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5517.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

Purpose: In vertebrates, production of visual chromophore relies on specific enzymes of which lecithin:retinol acyltransferase (LRAT) is a key component. LRAT, a representative of N1pC/P60 protein family is solely responsible for retinyl ester formation in the eye providing the necessary substrate for RPE65-dependent production of cis retinoids. Although the importance of LRAT in vision is clear, our understanding of its action is limited. The recently identified LRAT-specific domain which determines substrate specificity enables a new experimental approach to study this enzyme. Thus, the purpose of this work was to identify the key factors that govern enzymatic transfer of an acyl moiety and retinyl esters formation.

 
Methods
 

Methods: To compare the active site architectures of phospholipases and acyltransferases, high-resolution structures of HRASLS3, HRASLS3/LRAT chimeric protein or its catalytically impaired mutants were obtained using X-ray crystallography. The stability of the transient thioester catalytic intermediate was examined by mass spectrometry and correlated with accessibility of the active site for water molecules, which was probed by radiolytic footprinting methodology.

 
Results
 

Results: The LRAT-specific domain differentiated LRAT from related HRASLS proteins by inducing a structural reorganization associated with domain-swapping dimerization. This rearrangement was associated with slower decay of the thioester catalytic intermediate and thus contributed to effective acyl transferase activity. Neither structural changes within the oxyanion hole nor the side chains affecting activation of a nucleophilic water molecule contributed to the impaired thiester hydrolysis. Instead, limited access of water molecules to the active site is the key factor that determined effective inhibition of thioester hydrolysis and permitted use of vitamin A as an acyl acceptor.

 
Conclusions
 

Conclusion: Analyses of the HRASLS3/LRAT chimeric protein revealed valuable insights into the molecular adaptations that lead to acquisition of novel substrate specificity by modifying existing structural motifs. The crystallographic data reported herein represent the first structural data that shed light on the molecular architecture of LRAT and provide evidence for the functional significance of domain swapping dimerization.

 
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