Periplasmic nitrate reductase (Nap), a molybdenum (Mo) enzyme, catalyzes the reduction of nitrate (NO3-) to nitrite (NO2-) in the periplasm, and yet the physiological role of Nap is not completely clear. It has been suggested that nitrate reduction may be involved in redox homeostasis, nitrate scavenging, nitrate respiration, and perhaps in pathogenicity. We present for the first time the heterologous overexpression of NapA from a pathogenic organism, Campylobacter jejuni. NapA, contains a molybdopterin cofactor where Mo is coordinated by two pyranopterin molecules and a cysteine residue. We hypothesize nitrate binds tightly to the Mo center of C. jejuni NapA due to the physiologically low nitrate concentrations found in the gut where C. jejuni colonizes. There are over 50 known molybdopterin enzymes with similar molybdenum cofactors yet they have different specificities for substrates and catalyze diverse reactions. Molybdopterin enzymes vary in the first coordination sphere of the Mo active site specifically the Mo coordinating residue. The coordinating residue has been hypothesized to tune the redox potential shifting substrate preference. For example, DMSO reductase that reduces DMSO to DMS and TMAO reductase that reduces TMAO to TMA have molybdenum centers coordinated by serine instead of cysteine. In order to study the tuning properties of the coordinating residue, recombinant native NapA and coordinating residue variants of NapA have been expressed. The kinetic properties of the native enzyme and the coordinating residue variants will be explored.

Exchanging protein ligands to examine the impact of molybdenum coordination in periplasmic nitrate reductase from Campylobacter jejuni

2019 Spring National Meeting

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