RESUMO
In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Chlamydomonas reinhardtii , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , NADP , Oxirredução , Oxirredutases/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismoRESUMO
Viral accessory proteins of the human immunodeficiency virus (HIV), including virus protein R (Vpr), are crucial for the efficient replication of the virus in the host organism. While functional data are available for HIV-1 Vpr, there is a paucity of data describing the function and structure of HIV-2 Vpr. In this report, the construction of a His6-MBP-intein1-Vpr-intein2-Cyt b5-His6 fusion protein is presented. Unlike previous research efforts where only microgram quantities of HIV-1 Vpr could be produced, this construct enabled soluble milligram yields via an Escherichia coli over-expression system. Straightforward protein purification of HIV-2 Vpr was achieved by standard chromatography routines and autocatalytic intein cleavage. Preliminary structural studies by circular dichroism (CD) and NMR spectroscopy revealed that the protein is stable in the presence of micellar concentrations of the detergent DPC and adopts an α-helix secondary structure.
Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/isolamento & purificação , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/genética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilcolina/análogos & derivados , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.
