Two New Unspecific Peroxygenases from Heterologous Expression of Fungal Genes in Escherichia coli.

Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds.IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.

Fatty Acid Chain Shortening by a Fungal Peroxygenase.

A recently discovered peroxygenase from the fungus Marasmius rotula (MroUPO) is able to catalyze the progressive one-carbon shortening of medium and long-chain mono- and dicarboxylic acids by itself alone, in the presence of H2 O2 . The mechanism, analyzed using H218 O2 , starts with an α-oxidation catalyzed by MroUPO generating an α-hydroxy acid, which is further oxidized by the enzyme to a reactive α-keto intermediate whose decarboxylation yields the one-carbon shorter fatty acid. Compared with the previously characterized peroxygenase of Agrocybe aegerita, a wider heme access channel, enabling fatty acid positioning with the carboxylic end near the heme cofactor (as seen in one of the crystal structures available) could be at the origin of the unique ability of MroUPO shortening carboxylic acid chains.

From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase.

A new heme-thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position-a challenging reaction in organic chemistry-with H2 O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC-MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of (18) O from the cosubstrate H2 (18) O2 , demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids.

Impact of high-risk and low-risk human papillomavirus infections on the male genital tract: effects on semen inflammation and sperm quality.

Human Papillomavirus (HPV), a prevalent sexually transmitted infection, comprises high-risk (HR-HPV) and low-risk (LR-HPV) viruses, the former posing a high risk for developing malignancies whereas the latter mainly for benign warts. Despite increasing awareness of HPV's impact on men's health, the influence of HR-HPV and LR-HPV urogenital infections on male fertility potential remains uncertain. This study aimed to investigate whether male urogenital infection with HR- or LR-HPV associates with impaired sperm quality, oxidative stress, and inflammation. A total of 205 male patients attending an urology clinic were enrolled. Semen samples were analyzed for HPV using PCR and genotyped by RFLP. Semen quality was evaluated following WHO guidelines. Semen leukocytes, reactive oxygen species (ROS), and sperm viability were analyzed using flow cytometry. HPV was detected in 19% (39/205) of semen samples. HR-HPV infections were more prevalent, with HPV-16 being the most frequent genotype. Neither HR-HPV nor LR-HPV were associated with significant alterations in routine sperm quality parameters. However, HR-HPV+ individuals showed significantly higher levels of sperm necrosis and exhibited increased proportions of ROS+ spermatozoa compared to LR-HPV+ or control individuals. Furthermore, no significant semen inflammation was detected in patients infected with either HR-HPV or LR-HPV, and unexpectedly reduced semen leukocytes and inflammatory cytokines (IL-6 and IL-1ß) were observed in HR-HPV+ patients compared to controls. These observations underscore the importance of comprehensive HPV screening, including genotyping, in urology and fertility clinics to understand the progression of the infection, potential adverse effects on reproductive health, and the oncogenic risks involved.

Novel Fatty Acid Chain-Shortening by Fungal Peroxygenases Yielding 2C-Shorter Dicarboxylic Acids.

Unspecific peroxygenases (UPOs), the extracellular enzymes capable of oxygenating a potpourri of aliphatic and aromatic substrates with a peroxide as co-substrate, come out with a new reaction: carbon-chain shortening during the conversion of fatty acids with the well-known UPOs from Coprinopsis cinerea (rCciUPO) and Cyclocybe (Agrocybe) aegerita (AaeUPO). Although a pathway (Cα-oxidation) for shortening the hydrocarbon chain of saturated fatty acids has already been reported for the UPO from Marasmius rotula (MroUPO), it turned out that rCciUPO and AaeUPO shorten the chain length of both saturated and unsaturated fatty acids in a different way. Thus, the reaction sequence does not necessarily start at the Cα-carbon (adjacent to the carboxyl group), as in the case of MroUPO, but proceeds through the subterminal (ω-1 and ω-2) carbons of the chain via several oxygenations. This new type of shortening leads to the formation of a dicarboxylic fatty acid reduced in size by two carbon atoms in the first step, which can subsequently be further shortened, carbon by carbon, by the UPO Cα-oxidation mechanism.

Advances in enzymatic oxyfunctionalization of aliphatic compounds.

Selective oxyfunctionalizations of aliphatic compounds are difficult chemical reactions, where enzymes can play an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as "dream biocatalysts" of great biotechnological interest because they catalyze the oxyfunctionalization of aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxyfunctionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible for their activities, emphasizing the differences found between well-known P450s and the novel fungal peroxygenases.