DyP-type peroxidases: a promising and versatile class of enzymes.

DyP peroxidases comprise a novel superfamily of heme-containing peroxidases, which is unrelated to the superfamilies of plant and animal peroxidases. These enzymes have so far been identified in the genomes of fungi, bacteria, as well as archaea, although their physiological function is still unclear. DyPs are bifunctional enzymes displaying not only oxidative activity but also hydrolytic activity.

A generic, whole-cell-based screening method for Baeyer-Villiger monooxygenases.

Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format.

Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B.

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli.

Functionalization of oxidases with peroxidase activity creates oxiperoxidases: a new breed of hybrid enzyme capable of cascade chemistry.

The covalent flavoprotein alditol oxidase (AldO) from Streptomyces coelicolor A3(2) was endowed with an extra catalytic functionality by fusing it to a microperoxidase. Purification of the construct resulted in the isolation of a synthetic bifunctional enzyme that was both fully covalently flavinylated and heminylated: an oxiperoxidase. Characterization revealed that both oxidase and peroxidase functionalities were active, with the construct functioning as a single-component xylitol biosensor.

Decorating microbes: surface display of proteins on Escherichia coli.

Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins.

A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily.

DyP-type peroxidases comprise a novel superfamily of heme-containing peroxidases which is unrelated to the superfamilies of known peroxidases and of which only a few members have been characterized in some detail. Here, we report the identification and characterization of a DyP-type peroxidase (TfuDyP) from the thermophilic actinomycete Thermobifida fusca. Biochemical characterization of the recombinant enzyme showed that it is a monomeric, heme-containing, thermostable, and Tat-dependently exported peroxidase.

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis.

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps.

Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins.

Little is known about the quality control of proteins upon integration in the inner membrane of Escherichia coli. Here, we demonstrate that YidC and FtsH are adjacent to a nascent, truncated membrane protein using in vitro photo cross-linking. YidC plays a critical but poorly understood role in the biogenesis of E.

Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC.

Members of the YidC/Oxa1/Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Escherichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in concert with the Sec-translocon and as a separate insertase independent of the translocon.

Flexibility in targeting and insertion during bacterial membrane protein biogenesis.

The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E.

Contribution of the FtsQ transmembrane segment to localization to the cell division site.

The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane.

Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA.

Inner membrane proteins (IMPs) of Escherichia coli use different pathways for membrane targeting and integration. YidC plays an essential but poorly defined role in the integration and folding of IMPs both in conjunction with the Sec translocon and as a Sec-independent insertase. Depletion of YidC only marginally affects the insertion of Sec-dependent IMPs, whereas it blocks the insertion of a subset of Sec-independent IMPs.

The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential.

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively.

F(1)F(0) ATP synthase subunit c is targeted by the SRP to YidC in the E. coli inner membrane.

Escherichia coli inner membrane proteins (IMPs) use different pathways for targeting and membrane integration. We have examined the biogenesis of the F1F0 ATP synthase subunit c, a small double spanning IMP, using complementary in vivo and in vitro approaches. The data suggest that F0c is targeted by the SRP to the membrane, where it inserts at YidC in a Sec-independent mechanism.