Structural Basis of Biological Nitrile Reduction

Chikwana, Vimbai M.; Stec, Boguslaw; Lee, Bobby W.K.; Crécy‐Lagard, Valérie de; Iwata‐Reuyl, Dirk; Swairjo, Manal A.

doi:10.1074/jbc.m112.388538

Cited by 29 publications

(79 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Multiple monomers come together to form a barrel and two barrels combine head on to form the final multimeric tunnel 9 . The number of individual domains varies within the T-fold family, and size exclusion studies and X-ray crystal structures have shown QueF to be a homodecamer 10,8 . …”

Section: Quefmentioning

confidence: 99%

Cofactor binding determinants in the NADPH-dependent nitrile-oxidoreductase QueF

Cohen¹

2016

View full text Add to dashboard Cite

The NADPH-dependent nitrile-oxidioreductase, QueF is the only known enzyme capable of reducing a nitrile group to an amine. This ability makes it an attractive alternative to conventional industrial nitrile redution. Understating how QueF binds NADPH may lead to the development of an enzyme capable of using the less expensive reductive cofactor NADH. A cofactor docking model indicates that key residues, Q60 and Y21, interact with the ribose phosphate moiety unique to NADPH. Mutants of these residues (Q60A, Q60N, Q60E, Y21A and Y21F) were developed for steady-state kinetic analysis. Modification to either residue resulted in a decrease in binding and catalytic activity when using NADPH as a hydride source. Q60E had the greatest reduction in activity (0.45% of WT). There appears to be both charge and steric factors involved in direct cofactor binding. Neither WT or mutant enzymes were able to utilize NADH as a reductive cofactor to any observable extent.

show abstract

Section: Quefmentioning

confidence: 99%

Cofactor binding determinants in the NADPH-dependent nitrile-oxidoreductase QueF

Cohen¹

2016

View full text Add to dashboard Cite

show abstract

“…QueF among all other enzymes of the Q pathway, therefore, presents a potential target for antiinfective strategies directed selectively against bacteria. QueF also has attracted considerable mechanistic interest (3,(5)(6)(7)17), for the reaction catalyzed by it is apparently unique to biology.…”

Section: Introductionmentioning

confidence: 99%

“…Two-fold enzymatic reduction of preQ 0 is proposed to proceed in three catalytic steps, as shown in Scheme 1 (3)(4)(5)(6)(7). A covalent thioimide adduct is formed between the enzyme's catalytic nucleophile (cysteine) and preQ 0 (3,5,7).…”

Section: Introductionmentioning

confidence: 99%

“…Mobile elements of the protein structure are often used to close down over the substrate while it undergoes the catalytic transformation into product. The nitrile reductase QueF deals with a notably difficult problem of intermediate sequestration (3)(4)(5)(6)(7). QueF catalyzes a four-electron reduction of a nitrile to an amine in two NADPHdependent steps, presumably via an imine intermediate.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, to ensure that every substrate "makes it through" to the amine, QueF must do both, keep a firm grip on the labile imine during the catalytic act and exclude water from the binding site. This study was performed to identify elements of the QueF catalytic apparatus that make nitrile reductases the high-fidelity enzymes they are by nature, converting nitrile to amine without detectable loss of the supposed imine to off-pathway reactions (3)(4)(5)(6)(7).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase QueF from Escherichia coli

Jung

Nidetzky

2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

In the biosynthesis of the tRNA-inserted nucleoside queuosine, the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine (preQ 1 ), a biologically unique four-electron reduction of a nitrile to an amine. The QueF mechanism involves a covalent thioimide adduct between the enzyme and preQ 0 which undergoes reduction to preQ 1 in two NADPH-dependent steps, presumably via an imine intermediate. Protecting a labile imine from interception by water is fundamental to QueF catalysis for proper enzyme function. In the QueF from E. coli, the conserved Glu 89 and Phe 228 residues together with a mobile structural element comprising the catalytic Cys 190 form a substratebinding pocket that secludes the bound preQ 0 completely from solvent. We show here that residue substitutions (E89A, E89L, F228A) targeted at opening up the binding pocket weakened preQ 0 binding at the preadduct stage, by up to +10 kJ/mol, and profoundly affected on catalysis. Unlike wildtype enzyme, the QueF variants, including L191A and I192A, were no longer selective for preQ 1 formation. The E89A, E89L and F228A variants performed primarily (≥ 90%) a two-electron reduction of preQ 0 , releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product. The preQ 0 reduction by L191A and I192A gave preQ 1 and 7-formyl-7-deazaguanine at a 4:1 and 1:1 ratio, respectively. The proportion of 7-formyl-7-deazaguanine in total product increased with increasing substrate concentration, suggesting a role for preQ 0 in a competitor-induced release of the imine intermediate. Collectively, these results provide direct evidence for the intermediacy of an imine in the QueF-catalyzed reaction. They reveal determinants of QueF structure required for imine sequestration and hence for a complete nitrile-toamine conversion by this class of enzymes.

show abstract

A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase

Zhou

et al. 2018

ChemBioChem

View full text Add to dashboard Cite

Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.

show abstract

Structural Basis of Biological Nitrile Reduction

Cited by 29 publications

References 23 publications

Cofactor binding determinants in the NADPH-dependent nitrile-oxidoreductase QueF

Cofactor binding determinants in the NADPH-dependent nitrile-oxidoreductase QueF

Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase QueF from Escherichia coli

A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase

Contact Info

Product

Resources

About