Monooxygenase Puns

Journal of the American Chemical SocietyDOI: 10.1021/jacs.1c01180

Christine E. Schulz, Rebeca G. Castillo, Dimitrios A. Pantazis, Serena DeBeer, and Frank Neese

https://ift.tt/3sI4AV9

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β‐position but LCA is exclusively hydroxylated at the 6β‐position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity‐determining residues. Alanine scanning identified S240A as a UDCA‐producing variant. A synthetic 'small but smart' library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio‐ and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10‐fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.

https://ift.tt/3jgY42L

The interest in indole dearomatization, which serves as a useful tool in the total synthesis of related alkaloid natural products, has recently been renewed with the intention of developing new methods efficient in both yield and stereoselective control. Here, we report an enzymatic approach for the oxidative dearomatization of indoles in the asymmetric synthesis of a variety of furoindolines with a vicinal quaternary carbon stereogenic center. This approach depends on the activity of a flavin‐dependent monooxygenase, TsrE, which is involved in the biosynthesis of bicyclic thiopeptide antibiotic thiostrepton. TsrE catalyzes 2,3‐epoxidation and subsequent epoxide opening in a highly enantioselective manner during the conversion of 2‐methyl‐indole‐3‐acetic acid or 2‐methyl‐tryptophol to furoindoline , with up to > 99% conversion and > 99% ee under mild reaction conditions. Complementing current chemical methods for oxidative indole dearomatization, the TsrE activity‐based approach enriches the toolbox in the asymmetric synthesis of products possessing a furoindoline skeleton.

https://ift.tt/39lvj32

Journal of the American Chemical SocietyDOI: 10.1021/jacs.0c11920

Xuanyu Feng, Yang Song, Justin S. Chen, Ziwan Xu, Soren J. Dunn, and Wenbin Lin

https://ift.tt/3oqVZ7I

The interprotein electron‐transfer (ET) mechanism between lytic polysaccharide monooxygenases (LPMOs) and cellobiose dehydrogenase (CDH) was deciphered by multiscale simulations. Our simulations show that I) the specific ET pathway involves water molecules; II) the long‐range ET is enhanced by exothermic oxygen binding; III) enhanced ET is spin‐regulated and favors the quartet state over the doublet state.

Abstract

Long‐range electron transfer (ET) in metalloenzymes is a general and fundamental process governing O2 activation and reduction. Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the oxidative cleavage of insoluble polysaccharides, but their reduction mechanism by cellobiose dehydrogenase (CDH), one of the most commonly used enzymatic electron donors, via long‐range ET is still an enigma. Using multiscale simulations, we reveal that interprotein ET between CDH and LPMO is mediated by the heme propionates of CDH and solvent waters. We also show that oxygen binding to the copper center of LPMO is coupled with the long‐range interprotein ET. This process, which is spin‐regulated and enhanced by the presence of O2, directly leads to LPMO−CuII−O2−, bypassing the formation of the generally assumed LPMO−CuI species. The uncovered ET mechanism rationalizes experimental observations and might have far‐reaching implications for LPMO catalysis as well as the O2‐ or CO‐binding‐enhanced long‐range ET processes in other metalloenzymes.

https://ift.tt/2ISToDH

Journal of the American Chemical SocietyDOI: 10.1021/jacs.9b13756

https://ift.tt/2QrwqnY

Journal of the American Chemical SocietyDOI: 10.1021/jacs.0c02724

https://ift.tt/3bejTvJ

Journal of the American Chemical SocietyDOI: 10.1021/jacs.0c05613

https://ift.tt/3iby7kX

One mutant, six targets : The regio‐ and stereoselective C−H activation for hydroxylation of six different steroids with formation of C7β alcohols was accomplished using a single P450 mutant evolved by protein library construction based on a special DNA assembly and cloning procedure. The C7β steroidal alcohols, not readily accessible by synthetic reagents or catalysts, are of intense interest as therapeutic drugs.

Abstract

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity.

https://ift.tt/2JDNtiZ

Aliphatic synthetic intermediates with high added value are generally produced from alkane sources (e.g., petroleum) by inert carbon‐hydrogen (C‐H) bond activation using classical chemical methods (high temperature, rare metals). As an alternative approach for these reactions, alkane monooxygenase from Pseudomonas putida (alkB) is able to catalyze the difficult terminal oxyfunctionalization of alkanes selectively and under mild conditions. Here, we report an electrosynthetic system using an alkB biocathode which produces alcohols, epoxides, and sulfoxides through bioelectrochemical hydroxylation, epoxidation, sulfoxidation, and demethylation. The capacity of the alkB binding pocket to protect internal functional groups, a lucrative capability, is also demonstrated. By coupling our alkB biocathode with a hydrogenase bioanode and using H2 as a clean fuel source, we have developed and characterized a series of enzymatic fuel cells (EFCs) capable of oxyfunctionalization while simultaneously producing electricity.

https://ift.tt/2wtgz1v

I don't want to step on anybody's toes here, but the amount of non-dad jokes here in this subreddit really annoys me. First of all, dad jokes CAN be NSFW, it clearly says so in the sub rules. Secondly, it doesn't automatically make it a dad joke if it's from a conversation between you and your child. Most importantly, the jokes that your CHILDREN tell YOU are not dad jokes. The point of a dad joke is that it's so cheesy only a dad who's trying to be funny would make such a joke. That's it. They are stupid plays on words, lame puns and so on. There has to be a clever pun or wordplay for it to be considered a dad joke.

Again, to all the fellow dads, I apologise if I'm sounding too harsh. But I just needed to get it off my chest.

Do your worst!

I'm surprised it hasn't decade.

For context I'm a Refuse Driver (Garbage man) & today I was on food waste. After I'd tipped I was checking the wagon for any defects when I spotted a lone pea balanced on the lifts.

I said "hey look, an escaPEA"

No one near me but it didn't half make me laugh for a good hour or so!

Edit: I can't believe how much this has blown up. Thank you everyone I've had a blast reading through the replies 😂

It really does, I swear!

They’re on standbi

Buenosdillas

We report engineering of a P450 monooxygenase for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid to produce ursodeoxycholic acid (UDCA). Structural and 3DM analysis, and molecular docking, identified selectivity‐influencing residues. A “small but smart” mutant library was then screened with a selective colorimetric assay. The best mutant has nearly perfect regio‐ and stereoselectivity, enabling a new route for UDCA synthesis.

Abstract

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β‐position but LCA is exclusively hydroxylated at the 6β‐position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity‐determining residues. Alanine scanning identified S240A as a UDCA‐producing variant. A synthetic “small but smart” library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio‐ and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10‐fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.

https://ift.tt/3jgY42L

Long‐range electron transfer (ET) in metalloenzymes is a general and fundamental process governing activation and O 2 reduction. Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the oxidative cleavage of insoluble polysaccharides, but their reduction mechanism by cellobiose dehydrogenase (CDH), one of the most used enzymatic electron donors, via long‐range ET, is still an enigma. Using multiscale simulations, we reveal that interprotein ET between CDH and LPMO is mediated by the heme propionates of CDH and solvent waters. Interestingly, we find that oxygen binding to the copper center of LPMO is coupled with the long‐range interprotein ET. This process, which is spin‐regulated and enhanced by the presence of O 2 , directly leads to LPMO‐Cu(II)‐O 2 • , bypassing formation of the generally suggested LPMO‐Cu(I) species. The uncovered ET mechanism rationalizes experimental observations and has far‐reaching implications in LPMO catalysis, as well as the O 2 or CO binding‐enhanced long‐range ET processes in other metalloenzymes.

https://ift.tt/2ISToDH

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma and cerebral ischemia. Since position C7 is spatially far away from any functional groups that could direct C–H activation, these transformations are not readily possible using modern synthetic organic techniques. We report P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7‐regio‐ and β‐stereoselectivity as well as high activity. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity.

https://ift.tt/2JDNtiZ