Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

Abstract

The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo–MGD) cofactor and one [4Fe–4S] iron–sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (NapSgel) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (NapSput) was examined at varied temperature. Irreversible deactivation of NapSgel and NapSput occurred at 54.5 and 65°C, respectively. When NapSgel was preincubated at 21–70°C for 30min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54°C, which suggested that NapSgel was poised for optimal catalysis at modest temperatures and, unlike NapSput, did not benefit from thermally-induced refolding. At 20°C, NapSgel reduced selenate at 16% of the rate of nitrate reduction. NapSput did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in NapSgel that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the NapSgel cold-adapted phenotype. Protein homology modeling of NapSgel using a mesophilic template with 66% amino acid identity showed the majority of substitutions occurred at the protein surface distal to the Mo–MGD cofactor. Two mesophilic↔psychrophilic substitutions (Asn↔His, Val↔Trp) occurred in a region close to the surface of the NapA substrate funnel resulting in potential interdomain π–π and/or cation–π interactions. Three mesophilic↔psychrophilic substitutions occurred within 4.5Å of the Mo–MGD cofactor (Phe↔Met, Ala↔Ser, Ser↔Thr) resulting in local regions that varied in hydrophobicity and hydrogen bonding networks. These results contribute to the understanding of thermal protein adaptation in a redox-active mononuclear molybdenum enzyme and have implications in optimizing the design of low-temperature environmental biosensors.