Identification and characterization of 2‐naphthoyl‐coenzyme A reductase, the prototype of a novel class of dearomatizing reductases

Abstract

SummaryThe enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoylcoenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin-containing 'old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases.

Hydroxyl regioisomerization of anthracycline catalyzed by a four-enzyme cascade

“…It is promising that this dehydroxylation could be used to provide aimed anthracycline analogs in the future by protein engineering. In addition, dearomatizations are chemically challenging in a sense both of chemistry and of biology (16,17,27), as the asymmetric hydrogenation of aromatic compounds, which was considered a versatile and practical synthetic strategy method to obtain chiral compound, has been an active and attractive field of methodology research (28,29). Recently, characterization of the NADPH-dependent reductases involved in fungal 1,8-dihydroxynaphthalene melanin, monodictyphenone, and aflatoxin B1 biosynthesis expanded the substrates to hydroxynaphthoquinones and tricyclic anthrahydroquinones (30)(31)(32)(33).…”

Anaerobic Degradation of Benzene and Polycyclic Aromatic Hydrocarbons

“…Surprisingly, none of these reductions were ATP independent, and, unlike the class I and II benzoyl-CoA reductases, the enzymes were not sensitive to oxygen. Eberlein et al [2013a] purified the 2-naphthoyl-CoA reductase from strain N47 and identified the encoding gene N47_638220, referred to as ncr . The 2-naphthoylCoA reductase is composed of two 73.3-kDa subunits encoded by gene N47_G38220 forming an α 2 -homo dimer of 156 kDa.…”

“…The protein sequence showed high similarities to members of the flavin-containing 'old yellow enzyme' family, which uses NADPH as cofactor [Sawers, 2015]. Genes within proximity of ncr in the genome are annotated as oxidoreductases, and Eberlein et al [2013a] proposed their function to be electron transfer proteins from a nonidentified electron donor to 2-naphthoyl-CoA reductase. The 2-naphthoyl-CoA reductase assay catalyzed a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA.…”

“…Members of the class I benzoyl-CoA reductase of Azoarcus evansii and T. aromatica usually depend on reduced ferredoxin or NADPH coupled to a NADPH:ferredoxin oxidoreductase as electron donor Ebenau-Jehle et al, 2003;Eberlein et al, 2013a]. Furthermore, the use of the strong reducing agent Ti(III) citrate did not enhance the activity of the 5,6,7,8-tetrahydro-2-naphthoyl-CoA as seen with the class I benzoyl-CoA reductases [Boll and Fuchs, 1995].…”

Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase

“…the sulfate-reducing Deltaproteobacterium NaphS2 or the enrichment culture N47. During growth of these obligate anaerobes with naphthalene, the 2-naphthoyl-CoA intermediate is first reduced to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA) by two flavin-dependent reductases belonging to the old yellow enzyme family [Eberlein et al, 2013a;Estelmann et al, 2015]. These enzymes can be regarded as a third class of dearomatizing aryl-CoA reductases .…”