The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis

We describe here the molecular cloning and analysis of the M r 14,000 and 16,000 outer arm dynein light chains (DLCs) from Chlamydomonas flagella. Within the outer arm, the M r 14,000 DLC apparently is associated with the intermediate chains at the base of the soluble dynein particle; the M r 16,000 DLC interacts directly with the ␣ dynein heavy chain. Sequence analysis indicates that both molecules are novel members of the thioredoxin superfamily and share ϳ30% sequence identity with thioredoxin from Penicillium. Both DLCs have a perfect copy of the thioredoxin active site (WCGPCK); the M r 16,000 DLC also contains the canonical P-loop motif (AX 4 GKS). There is a single gene for both DLCs within Chlamydomonas and only single messages that were upregulated more than 10-fold upon deflagellation were observed on Northern blots. Both recombinant DLCs were specifically eluted from a phenylarsine oxide matrix with ␤-mercaptoethanol indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction. Furthermore, we demonstrate that outer (but not inner) arm dynein may be purified on the basis of its affinity for phenylarsine oxide suggesting that the predicted redox-sensitive vicinal dithiols exist within the native complex.

Section: Resultsmentioning

confidence: 99%

Two Functional Thioredoxins Containing Redox-sensitive Vicinal Dithiols from the Chlamydomonas Outer Dynein Arm

Patel‐King

Benashski

Harrison

et al. 1996

“…The class includes prokaryotic and eukaryotic thioredoxin reductases (35,36) and bacterial PrxRs (37). The sequence is present in T. aquaticus PrxR between residues 337 and 357 (Fig.…”

Section: Cloning and Sequences Of Prxr And Prx Genes-mentioning

confidence: 99%

Cloning, Overexpression, and Characterization of Peroxiredoxin and NADH Peroxiredoxin Reductase from Thermus aquaticus

Logan

Mayhew

2000

The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus. prx is located upstream from prxR, the two genes being separated by 13 bases. The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria. The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues. PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension. The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers. The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer. PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide. The activity of Prx is much less than has been observed with homologous proteins. Prx appears to be inactivated by cumene hydroperoxide. PrxR itself has low peroxidase activity.

“…In microbial eukaryotes, ROIs are involved in development, cell differentiation (10), and host-pathogen interaction (11). Also, a possible role of oxidoreductase systems in the penicillin biosynthesis has been discussed for Penicillium chrysogenum and Streptomyces clavuligerus (12,13). In this report, we describe the isolation and characterization of a thioredoxin system from A. nidulans, which is an important model organism to study all kinds of biological questions, including development and the production of secondary metabolites (14).…”

mentioning

confidence: 99%

The Thioredoxin System of the Filamentous Fungus Aspergillus nidulans

Thön

Abdallah

Hortschansky

et al. 2007

100

Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase.Due to the metabolism of molecular oxygen as the final electron acceptor of the respiratory chain, all aerobic organisms are exposed to reactive oxygen intermediates (ROIs).2 Whereas low concentrations of ROI are supposed to function as secondary messengers, elevated ROI levels can lead to damage of biological macromolecules, like DNA, lipids, and proteins. However, there are several enzymatic and non-enzymatic defense mechanisms that are able to detoxify ROI efficiently. These mechanisms include superoxide dismutases, catalases, peroxidases, and the tripeptide glutathione. Glutathione is the most abundant intracellular thiol compound and serves as a powerful antioxidant and radical scavenger. Another important redox system is formed by the thioredoxin system. Thioredoxin systems are composed of two enzymes, i.e. thioredoxin (Trx) and thioredoxin reductase (TrxR) (1, 2). Thioredoxins are small, ubiquitously distributed proteins with a molecular mass of 12-13 kDa. Due to their redox-active cysteine pair in the active site (WCGPC), they are able to cycle between their oxidized disulfide (Trx-S 2 ) and reduced dithiol [Trx-(SH) 2 ] forms. In an NADPH-dependent protein disulfide reduction reaction TrxR catalyzes the reduction of oxidized thioredoxin using NADPH as electron donor, its own redox-active cysteine pair and FAD as cofactor. Reduced Trx directly reduces the disulfide in the target protein. This NADPH-dependent disulfide reductio...