Purification and Characterization of N2 Enzyme

Chemicals, biochemicals, buffers, and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), Fisher Scientific Inc. (Pittsburgh, PA), Fluka Chemical Corp. (Milwaukee, WI), or EMD Millipore, Inc. (Billerica, MA). 2-Hydroxymuconate (2-HM)¹¹ and 3-bromopropiolate (3-BP)¹² were synthesized by the indicated references. Phenylenolpyruvate (PP) was purchased from Fluka Chemical Corp. (Milwaukee, WI) as the crystalline free acid, which exists exclusively in the enol form. Propiolic acid (98%) was purchased from Sigma-Aldrich, and purified further prior to use by distillation. N2 was purified by a literature procedure.^{13 (link)} N2 is an enzyme from Gammaproteobacteria bacterium SG8_31 (UniProt accession A0A0S8FF56) and is in the 4-oxalocrotonate tautomerase (4-OT) subgroup, as described elsewhere.^{13 (link)} The DEAE Sephadex, Phenyl-Sepharose 6 Fast Flow, and Sephadex G-75 resins were obtained from GE Healthcare Bio-sciences (Pittsburgh, PA). The Econo-Column^® chromatography columns were obtained from BioRad (Hercules, CA). The Amicon stirred cell concentrators and the ultrafiltration membranes (3,000 or 5,000 Da, MW cutoff) were purchased from EMD Millipore Inc.