RESUMEN
The pathogenic bacteria Legionella pneumophila is known to cause Legionnaires' Disease, a severe pneumonia that can be fatal to immunocompromised individuals and the elderly. Shohdy et al. identified the L. pneumophila vacuole sorting inhibitory protein VipF as a putative N-acetyltransferase based on sequence homology. We have characterized the basic structural and functional properties of VipF to confirm this original functional assignment. Sequence conservation analysis indicates two putative CoA-binding regions within VipF. Homology modeling and small angle X-ray scattering suggest a monomeric, dual-domain structure joined by a flexible linker. Each domain contains the characteristic beta-splay motif found in many acetyltransferases, suggesting that VipF may contain two active sites. Docking experiments suggest reasonable acetyl-CoA binding locations within each beta-splay motif. Broad substrate screening indicated that VipF is capable of acetylating chloramphenicol and both domains are catalytically active. Given that chloramphenicol is not known to be N-acetylated, this is a surprising finding suggesting that VipF is capable of O-acetyltransferase activity. Proteins 2016; 84:1422-1430. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Acetilcoenzima A/química , Acetiltransferasas/química , Proteínas Bacterianas/química , Cloranfenicol/química , Legionella pneumophila/enzimología , Acetilcoenzima A/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cloranfenicol/metabolismo , Clonación Molecular , Secuencia Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Legionella pneumophila/química , Simulación de Dinámica Molecular , Plásmidos/química , Plásmidos/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Single nucleotide polymorphisms (SNPs) in DNA can result in phenotypes where the biochemical basis may not be clear due to the lack of protein structures. With the growing number of modeling and simulation software available on the internet, students can now participate in determining how small changes in genetic information impact cellular protein structure and function. We have developed a modular series of activities to engage lab or lecture students in examining the basis for common phenotypes. The activities range from basic phenotype testing/observation to DNA sequencing and simulation of protein structure and dynamics. We provide as an example study of the bitterness receptor TAS2R38 and PTC tasting, however these activities are applicable to other SNPs or genomic variants with a direct connection to an observable phenotype. These activities are modular and can be mixed to meet the student capabilities and infrastructure availability. The complete sequence of activities will demonstrate the direct connection between gene structure, protein structure and organism function. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):526-536, 2016.