Chemical modification of small molecules is a key step for the development of pharmaceuticals. S‐adenosyl‐l‐methionine (SAM) analogues are used by methyltransferases (MTs) to transfer alkyl, allyl ...and benzyl moieties chemo‐, stereo‐ and regioselectively onto nucleophilic substrates, enabling an enzymatic way for specific derivatisation of a wide range of molecules. l‐Methionine analogues are required for the synthesis of SAM analogues. Most of these are not commercially available. In nature, O‐acetyl‐l‐homoserine sulfhydrolases (OAHS) catalyse the synthesis of l‐methionine from O‐acetyl‐l‐homoserine or l‐homocysteine, and methyl mercaptan. Here, we investigated the substrate scope of ScOAHS from Saccharomyces cerevisiae for the production of l‐methionine analogues from l‐homocysteine and organic thiols. The promiscuous enzyme was used to synthesise nine different l‐methionine analogues with modifications on the thioether residue up to a conversion of 75 %. ScOAHS was combined with an established MT dependent three‐enzyme alkylation cascade, allowing transfer of in total seven moieties onto two MT substrates. For ethylation, conversion was nearly doubled with the new four‐enzyme cascade, indicating a beneficial effect of the in situ production of l‐methionine analogues with ScOAHS.
O‐acetyl‐l‐homoserine sulfhydrolase is used for the production of l‐methionine analogues from l‐homocysteine and off‐the shelf organic thiols. Combined with methyltransferase‐catalysed alkylation cascades, this enables the transfer of a wide range of moieties onto various substrates.
S‐Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM‐dependent enzymes of great interest in ...biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S‐adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one‐pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems’ flexibility is showcased by the transfer of an ethyl group with a cobalamin‐dependent radical SAM MT using S‐adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.
The biomimetic regeneration system for S‐adenosylmethionine (SAM) and SAM analogues presented is based on the salvage of the adenine moiety and in situ supply of d‐ribose and polyphosphate. It is compatible with a broad range of SAM‐dependent enzymes including aminopropyl transferases, and is shown to support ethylation reactions with both conventional and radical SAM methyltransferases.
Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far‐reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by ...allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M2+R), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M2+A) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M2+A affords the overall lowest energy complex, which drives the transition from M2+R to M2+A. While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.
Pyruvate dependent Class II aldolases require a ketoacid to induce a metal shift from resting state to active enzyme. This occurs without alterations in the protein structure and might be a common, but to date overlooked, feature in many metalloproteins.
CYTH proteins make up a large superfamily that is conserved in all three domains of life. These enzymes have a triphosphate tunnel metalloenzyme (TTM) fold, which typically results in phosphatase ...functions, e.g., RNA triphosphatase, inorganic polyphosphatase, or thiamine triphosphatase. Some CYTH orthologs cyclize nucleotide triphosphates to 3′,5′-cyclic nucleotides. So far, archaeal CYTH proteins have been annotated as adenylyl cyclases, although experimental evidence to support these annotations is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our in silico studies derived ten major subclasses within the CYTH family implying a close relationship between these archaeal CYTH enzymes and class IV adenylyl cyclases. However, initial biochemical characterization reveals inability of SaTTM to produce any cyclic nucleotides. Instead, our structural and functional analyses show a classical TTM behavior, i.e., triphosphatase activity, where pyrophosphate causes product inhibition. The Ca2+-inhibited Michaelis complex indicates a two-metal-ion reaction mechanism analogous to other TTMs. Cocrystal structures of SaTTM further reveal conformational dynamics in SaTTM that suggest feedback inhibition in TTMs due to tunnel closure in the product state. These structural insights combined with further sequence similarity network–based in silico analyses provide a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.
Methylation reactions are of significant interest when generating pharmaceutically active molecules and building blocks for other applications. Synthetic methylating reagents are often toxic and ...unselective due to their high reactivity. S‐Adenosyl‐l‐methionine (SAM)‐dependent methyltransferases (MTs) present a chemoselective and environmentally friendly alternative. The anthranilate N‐MT from Ruta graveolens (RgANMT) is involved in acridone alkaloid biosynthesis, methylating anthranilate. Although it is known to methylate substrates only at the N‐position, the closest relatives with respect to amino acid sequence similarities of over 60 % are O‐MTs catalysing the methylation reaction of caffeate and derivatives containing only hydroxyl groups (CaOMTs). In this study, we investigated the substrate range of RgANMT and a CaOMT from Prunus persica (PpCaOMT) using compounds with both, an amino‐ and hydroxyl group (aminophenols) as possible methyl group acceptors. For both enzymes, the reaction was highly chemoselective. Furthermore, generating cofactor derivatives in situ enabled the transfer of other alkyl chains onto the aminophenols, leading to an enlarged pool of products. Selected MT reactions were performed at a preparative biocatalytic scale in in vitro and in vivo experiments resulting in yields of up to 62 %.
Chemoselective S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases (MTs) are a promising alternative to traditional synthetic methylation reactions. In presence of multiple nucleophiles, the enzymatic transfer of the carbon fragment is highly chemoselective for N‐ and O‐MTs. Besides methylation, the generation of SAM derivatives enables the transfer of altered groups onto the substrates increasing the pool of products.
Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far‐reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by ...allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M2+R), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M2+A) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M2+A affords the overall lowest energy complex, which drives the transition from M2+R to M2+A. While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.
Pyruvate dependent Class II aldolases require a ketoacid to induce a metal shift from resting state to active enzyme. This occurs without alterations in the protein structure and might be a common, but to date overlooked, feature in many metalloproteins.
In this randomized comparison of stenting and endarterectomy as treatment for carotid-artery stenosis, there was no significant difference in the rate of the composite primary end point of stroke, ...myocardial infarction, or death (7.2% and 6.8%, respectively; P=0.51). Stroke was more common with carotid-artery stenting than carotid endarterectomy; myocardial infarction was more common with carotid endarterectomy. The 4-year rate of stroke or death was 6.4% for carotid-artery stenting and 4.7% for carotid endarterectomy (P=0.03).
In this randomized comparison of stenting and endarterectomy as treatment for carotid-artery stenosis, there was no significant difference in the rate of the composite primary end point of stroke, myocardial infarction, or death (7.2% and 6.8%, respectively).
Carotid-artery atherosclerosis is an important cause of ischemic stroke.
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Carotid endarterectomy has been established as effective treatment for both symptomatic patients and asymptomatic patients.
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Carotid-artery stenting is another option for treatment. The results of randomized trials comparing carotid-artery stenting and carotid endarterectomy for use in symptomatic patients are conflicting.
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The primary aim of the Carotid Revascularization Endarterectomy vs. Stenting Trial (CREST) was to compare the outcomes of carotid-artery stenting with those of carotid endarterectomy among patients with symptomatic or asymptomatic extracranial carotid stenosis.
Methods
Study Design
CREST is a randomized, controlled trial with blinded end-point adjudication. Ethics review . . .
Stroke occurs more commonly after carotid artery stenting than after carotid endarterectomy. Details regarding stroke type, severity, and characteristics have not been reported previously. We ...describe the strokes that have occurred in the Carotid Revascularization Endarterectomy versus Stenting Trial (CREST).
CREST is a randomized, open-allocation, controlled trial with blinded end-point adjudication. Stroke was a component of the primary composite outcome. Patients who received their assigned treatment within 30 days of randomization were included. Stroke was adjudicated by a panel of board-certified vascular neurologists with secondary central review of clinically obtained brain images. Stroke type, laterality, timing, and outcome were reported. A periprocedural stroke occurred among 81 of the 2502 patients randomized and among 69 of the 2272 in the present analysis. Strokes were predominantly minor (81%, n=56), ischemic (90%, n=62), in the anterior circulation (94%, n=65), and ipsilateral to the treated artery (88%, n=61). There were 7 hemorrhages, which occurred 3 to 21 days after the procedure, and 5 were fatal. Major stroke occurred in 13 (0.6%) of the 2272 patients. The estimated 4-year mortality after stroke was 21.1% compared with 11.6% for those without stroke. The adjusted risk of death at 4 years was higher after periprocedural stroke (hazard ratio, 2.78; 95% confidence interval, 1.63-4.76).
Stroke, particularly severe stroke, was uncommon after carotid intervention in CREST, but stroke was associated with significant morbidity and was independently associated with a nearly 3-fold increased future mortality. The delayed timing of major and hemorrhagic stroke after revascularization suggests that these strokes may be preventable.
Aortic stenosis is a frequent valvular disease especially in elderly patients. Catheter-based valve implantation has emerged as a valuable treatment approach for these patients being either at very ...high risk for conventional surgery or even deemed inoperable. The German Aortic Valve Registry (GARY) provides data on conventional and catheter-based aortic procedures on an all-comers basis.
A total of 13 860 consecutive patients undergoing repair for aortic valve disease conventional surgery and transvascular (TV) or transapical (TA) catheter-based techniques have been enrolled in this registry during 2011 and baseline, procedural, and outcome data have been acquired. The registry summarizes the results of 6523 conventional aortic valve replacements without (AVR) and 3464 with concomitant coronary bypass surgery (AVR + CABG) as well as 2695 TV AVI and 1181 TA interventions (TA AVI). Patients undergoing catheter-based techniques were significantly older and had higher risk profiles. The stroke rate was low in all groups with 1.3% (AVR), 1.9% (AVR + CABG), 1.7% (TV AVI), and 2.3% (TA AVI). The in-hospital mortality was 2.1% (AVR) and 4.5% (AVR + CABG) for patients undergoing conventional surgery, and 5.1% (TV AVI) and AVI 7.7% (TA AVI).
The in-hospital outcome results of this registry show that conventional surgery yields excellent results in all risk groups and that catheter-based aortic valve replacements is an alternative to conventional surgery in high risk and elderly patients.
In a test-negative case–control study involving 1482 vaccinated health care workers and 3449 controls, the BNT162b2 and mRNA-1273 SARS-CoV-2 vaccines were 88.8% and 96.3% effective, respectively, at ...preventing symptomatic Covid-19. Efficacy was similar in subgroups according to age (<50 or ≥50 years), racial and ethnic groups, underlying conditions, and various levels of patient contact.
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