ABSTRACT
DHHC protein acyltransferases (PATs) catalyze the palmitoylation of eukaryotic proteins through an enzymatic mechanism that remains largely unexplored. In this study we have combined genetic and biochemical approaches to examine the molecular mechanism of palmitate transfer of the yeast Ras PAT, which is composed of Erf2 and Erf4. The palmitoylation reaction consists of two steps; they are autopalmitoylation of the enzyme to create a palmitoyl-Erf2 intermediate followed by the transfer of the palmitoyl moiety to the Ras substrate. Palmitoyl-CoA serves as the palmitate donor. To elucidate the kinetic properties of the Erf2·Erf4 PAT, we have developed a coupled enzyme assay that monitors the turnover of the palmitoyl-enzyme species indirectly by measuring the rate of CoASH release. Mutational analysis indicates that the DHHC motif constitutes the catalytic core of the enzyme required for autopalmitoylation and palmitoyl transfer to the Ras2 substrate. In the absence of Ras2, the palmitoyl-Erf2·Erf4 complex undergoes a cycle of hydrolysis and re-palmitoylation, implying that in the presence of palmitoyl-CoA, the complex is autopalmitoylated and competent to transfer palmitate to a protein substrate.
Subject(s)
Acyltransferases/metabolism , Lipoylation/physiology , Membrane Proteins/metabolism , Mutation, Missense , Palmitoyl Coenzyme A/metabolism , Protein Processing, Post-Translational/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Acyltransferases/genetics , Amino Acid Motifs , Membrane Proteins/genetics , Palmitoyl Coenzyme A/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.
Subject(s)
Zearalenone/chemistry , Biocatalysis , Drug Design , Enzymes/metabolism , Lipase/metabolism , Structure-Activity Relationship , Zearalenone/biosynthesisABSTRACT
Versatile intermediates 12'-iodovinblastine, 12'-iodovincristine and 11'-iodovinorelbine were utilized as substrates for transition metal based chemistry which led to the preparation of novel analogues of the vinca alkaloids. The synthesis of key iodo intermediates, their transformation into final products, and the SAR based upon HeLa and MCF-7 cell toxicity assays is presented. Selected analogues 27 and 36 show promising anticancer activity in the P388 murine leukemia model.
Subject(s)
Antineoplastic Agents, Phytogenic/chemical synthesis , Vinblastine/analogs & derivatives , Vinca Alkaloids/chemical synthesis , Vinca Alkaloids/pharmacology , Vincristine/analogs & derivatives , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Disease Models, Animal , Drug Screening Assays, Antitumor , HeLa Cells , Humans , Leukemia P388 , Mice , Molecular Structure , Structure-Activity Relationship , Vinblastine/chemical synthesis , Vinblastine/chemistry , Vinblastine/pharmacology , Vinca Alkaloids/chemistry , Vincristine/chemical synthesis , Vincristine/chemistry , Vincristine/pharmacologyABSTRACT
Palmitoylation enhances membrane association and plays a role in the subcellular trafficking and signaling function of proteins. Unlike other forms of protein lipidation, such as prenylation and myristoylation, palmitoylation is reversible and can therefore play a regulatory role. Enzyme activities have recently been described in mammals and yeast that carry out the palmitoylation of protein substrates. Protein acyltransferases (PATs) transfer a palmitoyl moiety derived from palmitoyl-CoA to a free thiol of a substrate protein to create a labile thioester linkage. Biochemical characterization and kinetic analysis of this new family of enzymes requires methods to purify PATs and their substrates, as well as methods to assay PAT activity. We describe a series of methods using yeast and bacterial expression systems to study protein acyltransferases.
