Sequence Conservation Does Not Always Signify a Functional Imperative as Observed in the Nitroreductase Superfamily

Musila, Jonathan M.; Rokita, Steven E.

doi:10.1021/acs.biochem.2c00037

Cited by 4 publications

(1 citation statement)

References 50 publications

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“…2 and 3 ; Supplementary Method 1 ), which show that all NMN + first shell residues are highly conserved. However, studies have shown that caution should be taken in equating sequence consensus to functional essentiality 42 , especially when the desired function (i.e., NMN + -utilizing activity) has not been explored by evolution. Instead of bioinformatic analysis, we next relied on a structure-guided approach (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

et al. 2022

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Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.

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Section: Resultsmentioning

confidence: 99%

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

et al. 2022

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Polar Interactions between Substrate and Flavin Control Iodotyrosine Deiodinase Function

Lemen,

Rokita

2024

Biochemistry

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Biochemical, kinetic, and structural characterization of a Bacillus tequilensis nitroreductase

Russo,

Rozeboom,

Wijma

et al. 2024

The FEBS Journal

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Nitroreductases (NRs) are NAD(P)H‐dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell‐imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized. Within this group of enzymes, only few are capable of amine synthesis, which is a fundamental chemical transformation for the pharmaceutical, agricultural, and textile industries. Herein, we provide a comprehensive description of a recently discovered NR from Bacillus tequilensis, named BtNR. This enzyme has previously been demonstrated to have the capability to fully convert nitro aromatic and heterocyclic compounds to their respective primary amines. In this study, we determined its biochemical, kinetic and structural properties, including its apparent melting temperature (Tm) of 59 °C, broad pH activity range (from pH 3 to 10) and a notably low redox potential (−236 ± 1 mV) in comparison to other well‐known NRs. We also determined its steady‐state and pre‐steady‐state kinetic parameters, which are consistent with other NRs. Additionally, we elucidated the crystal structure of BtNR, which resembles the well‐characterized Escherichia coli oxygen‐insensitive NAD(P)H nitroreductase (NfsB), and investigated the substrate binding in its active site through docking and molecular dynamics studies with four nitro aromatic substrates. Guided by these structural analyses, we probed the functional roles of active site residues by site‐directed mutagenesis. Our findings provide valuable insights into the biochemical and structural properties of BtNR, as well as its potential applications in biotechnology.

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Sequence Conservation Does Not Always Signify a Functional Imperative as Observed in the Nitroreductase Superfamily

Cited by 4 publications

References 50 publications

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

Polar Interactions between Substrate and Flavin Control Iodotyrosine Deiodinase Function

Biochemical, kinetic, and structural characterization of a Bacillus tequilensis nitroreductase

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