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. Moreover, these enzymes are able to oxidize a variety of organic compounds of which some are poorly converted by established peroxidases, including dyes, β-carotene, and aromatic sulfides. Interestingly, accumulating evidence shows that microbial DyP peroxidases play a key role in the degradation of lignin. Owing to their unique properties, these enzymes are potentially interesting for a variety of biocatalytic applications. In this review, we deal with the biochemical and structural features of DyP-type peroxidases as well as their promising biotechnological potential.

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. TfuDyP is not only active as dye-decolorizing peroxidase as it also accepts phenolic compounds and aromatic sulfides. In fact, it is able to catalyze enantioselective sulfoxidations, a type of reaction that has not been reported before for DyP-type peroxidases. Site-directed mutagenesis was used to determine the role of two conserved residues. D242 is crucial for catalysis while H338 represents the proximal heme ligand and is essential for heme incorporation. A genome database analysis revealed that DyP-type peroxidases are frequently found in bacterial genomes while they are extremely rare in other organisms. Most of the bacterial homologs are potential cytosolic enzymes, suggesting metabolic roles different from dye degradation. In conclusion, the detailed biochemical characterization reported here contributes significantly to our understanding of these enzymes and further emphasizes their biotechnological potential.

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Escherichia coli inner membrane proteins (IMPs) use different pathways for targeting and membrane integration. We have examined the biogenesis of the F 1 F 0 ATP synthase subunit c, a small double spanning IMP, using complementary in vivo and in vitro approaches. The data suggest that F 0 c is targeted by the SRP to the membrane, where it inserts at YidC in a Secindependent mechanism. F 0 c appears to be the first natural substrate of this novel pathway.

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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. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Secassociated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.

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. coli inner membrane proteins (IMPs). FtsH functions as a membrane chaperone and protease. Furthermore, we show that FtsH and its modulator proteins HflK and HflC copurify with tagged YidC and, vice versa, that YidC copurifies with tagged FtsH. These results suggest that FtsH and YidC have a linked role in the quality control of IMPs. Structured summary:MINT-6478034: hflB (uniprotkb:P0AAI3) physically interacts (MI:0218) with yidC (uniprotkb:P25714), hflK (uniprotkb:P0ABC7), hflC (uniprotkb:P0ABC3) by cross-linking studies (MI:0030) MINT-6478363: yidC (uniprotkb:P25714) physically interacts (MI:0218) with lldD (uniprotkb:P33232), rplD (uniprotkb:P60723), yrbD (uniprotkb:P64604), rpsA (uniprotkb:P0AG67), aphA1 (uniprotkb: P00551), dacC (uniprotkb:P08506), rpsC (uniprotkb:P0A7V3), rpsD (uniprotkb:P0A7V8), rpsE (uniprotkb:P0A7W1), rpoA (uniprotkb:P0A7Z4), ompC (uniprotkb:P06996), ompA (uniprotkb:P0A910), atpA (uniprotkb:P0ABB0), atpD (uniprotkb: P0ABB4), adhE (uniprotkb:P0A9Q7), hflB (uniprotkb:P0AAI3), hflC (uniprotkb:P0ABC3), hflK (uniprotkb:P0ABC7), lacI (uniprotkb:P03023), gapA (uniprotkb:P0A9B2), rbsB (uniprotkb: P02925), sdhA (uniprotkb:P0AC41), rho (uniprotkb:P0AG30), udp (uniprotkb:P12758), nuoC (uniprotkb:P33599), treB (uniprotkb:P36672) and manX (uniprotkb:P69797) by cross-linking studies (MI:0030) MINT-6477988: yidC (uniprotkb:P25714) physically interacts (MI:0218) with hflB (uniprotkb:P0AAI3), hflK (uniprotkb:P0ABC7), hflC (uniprotkb:P0ABC3), secD (uniprotkb:P0AG90) and secG (uniprotkb:P0AG99) by cross-linking studies (MI:0030) MINT-6478012: yidC (uniprotkb:P25714) physically interacts (MI:0218) with hflC (uniprotkb:P0ABC3) and hflK (uniprotkb:P0ABC7) by cross-linking studies (MI:0030) Ó

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. To eliminate the need for enzyme purification, this study describes a wholecell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INPbased system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.