Modulating the Molybdenum Coordination Sphere of <i>Escherichia coli</i> Trimethylamine <i>N</i>-Oxide Reductase

Kaufmann, Paul; Duffus, Benjamin R.; Mitrova, Biljana; Iobbi‐Nivol, Chantal; Teutloff, Christian; Nimtz, Manfred; Jänsch, Lothar; Wollenberger, Ulla; Leimkühler, Silke

doi:10.1021/acs.biochem.7b01108

Cited by 23 publications

(37 citation statements)

References 58 publications

(136 reference statements)

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“…In a recent study, we reported on the direct electrocatalytic reaction of TorA and the in vitro reconstituted chimera of this enzyme in order to indicate the role of the first coordination sphere of the cofactor on its potential and its activity . TorA‐FDH was among the most active enzyme variants.…”

Section: Resultsmentioning

confidence: 99%

“…The larger variation between the sensors can be explained by differences in the oxygen tolerance of the TorA‐WT. It was already reported that a contact of the WT enzyme with oxygen can damage the cofactor and as a result lower the activity of the enzyme . Since the TorA‐FDH variant seems to be less susceptible to oxidative damage compared to the WT enzyme, during the handling procedure, it was selected for the construction of the biosensor for TMAO detection.…”

Section: Resultsmentioning

confidence: 99%

“…The purification of E. coli TorA, apo‐TorA and the reconstitution of apo‐TorA with the cofactor of R. capsulatus formate dehydrogenase were performed as described in Kaufmann et al,…”

Section: Methodsmentioning

confidence: 99%

“…reported the reconstitution of an active TMAO reductase from the apoform of the enzyme (apo‐TorA) with the cofactor from Rhodobacter capsulatus formate dehydrogenase (FDH) (Scheme ). Direct and mediated electrochemistry established that high electrocatalytic activities were achieved when the enzyme was either expressed and purified under anaerobic conditions (referred to as TorA‐WT) or reconstituted with the FDH cofactor ligating a terminal sulfido ligand at the molybdenum atom (referred to as TorA‐FDH) …”

Section: Introductionmentioning

confidence: 99%

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Trimethylamine N‐Oxide Electrochemical Biosensor with a Chimeric Enzyme

et al. 2018

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For the first time, an enzyme-based electrochemical biosensor system for determination of trimethylamine N-oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low-potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA-FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA-FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of À 0.8 V [vs. Ag/AgCl (1 M KCl)] under ambient air conditions thanks to an additional enzymatic O 2 -scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA-FDH. This biosensor exhibits a sensitivity of 14.16 nA/μM TMAO in a useful measuring range of 2-110 μM with a detection limit of LOD = 2.96 nM (S/N = 3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16 � 2 s, the biosensor is stable over prolonged daily measurements (n = 20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum.It can also catalyze other N-oxide compounds, like 4-methyl [a] B.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…The purification of E. coli TorA, apo‐TorA and the reconstitution of apo‐TorA with the cofactor of R. capsulatus formate dehydrogenase were performed as described in Kaufmann et al,…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Trimethylamine N‐Oxide Electrochemical Biosensor with a Chimeric Enzyme

et al. 2018

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“…In other DMSO-reductase family enzymes, a sulfido ligand (Mo=S) at the metal was found. 21,22,36,[43][44][45] Accordingly, Mo=O/S distances as well as…”

Section: Mo Site Structure In Ydhv From Xasmentioning

confidence: 99%

Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli

Reschke

Duffus

Schrapers³

et al. 2019

Biochemistry

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The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco) containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homologies to a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. Our studies showed that YdhV has a preference for molybdenum over tungsten as metal to be inserted into the MPT backbone. The cofactor was identified to be of a bis-Mo-MPT type, which represents a novel form of Moco that has not been found earlier in any molybdoenzyme. In-depth characterization of YdhV by X-ray absorption and EPR spectroscopy revealed that the bis-Mo-MPT cofactor in YdhV is redox active, while a bis-W-MPT cofactor is redox inactive. The bis-Mo-MPT and bis-W-MPT cofactors include metal sites with the metal centers binding the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors. 10). Accordingly, E. coli is able to assemble not only molybdenum-, but also tungstencontaining cofactors. The presence of a Mo/W containing bis-MPT cofactor in E. coli opens a new route of Moco biosynthesis and expands the canon of the versatile molybdenum and tungsten enzymes. AUTHOR INFORMATION Corresponding Authors

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Recent progress in the development of immobilized penicillin G acylase for chemical and industrial applications: A mini‐review

Mohammed

Zhou

et al. 2019

Polymers for Advanced Techs

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Immobilized penicillin G acylase (PGA) as an important industrial catalyst can catalyze penicillin G potassium (PG) to 6‐aminopenicillanic acid (6‐APA). 6‐APA is an important intermediate for semisynthetic penicillin drugs, which occupies a huge market space in the anti‐inflammatory field; as a result, immobilized PGA occupies a huge market space in the pharmaceutical field. However, at present, there are different degrees of defects in the preparation and production process of immobilized PGAs on the market because of the huge demand; therefore, the performance of immobilized PGA and its productivity will bring huge economic benefits to enterprises. Therefore, research on immobilized PGA has always been a focus. This review first introduces the source, classification, structure, and catalytic mechanism of PGA and then studies the development of immobilization methods, immobilized carriers, reaction media, enzyme activity regeneration, and reactors of immobilized PGA in recent years.

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Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamine N-Oxide Reductase

Cited by 23 publications

References 58 publications

Trimethylamine N‐Oxide Electrochemical Biosensor with a Chimeric Enzyme

Trimethylamine N‐Oxide Electrochemical Biosensor with a Chimeric Enzyme

Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli

Recent progress in the development of immobilized penicillin G acylase for chemical and industrial applications: A mini‐review

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