Tungsten (W) containing oxidoreductases (WORs) are a phylogenetically distinct and diverse family of pyranopterin cofactor containing enzymes with over 4,000 representatives that can be subdivided into 92 distinct clades. Of the 92 clades, 24 have representatives found within the human gut microbiota and only one of the 24 clades has characterized representatives. Eubacterium limosum, an abundant gut microorganism, has two WORs, ElWOR1 (clade 87) and ElWOR2 (clade 81) encoded in its genome, together with a tungstate-specific transporter, Tup. E. limosum grows on lactate and produces the short chain fatty acids (SCFAs) butyrate and acetate which are important for human health. When growing on lactate, E. limosum accumulates more W than when growing on glucose and the genes expressing an electron bifurcating ElWOR1 and a W-containing formate dehydrogenase (FDH) are upregulated. W-pyranopterin cofactor dependent riboswitches also have a lactate-dependent regulation and intracellular acetaldehyde concentrations are significantly higher in lactate-grown cells. ElWOR1 is proposed to be involved in the detoxification of acetaldehyde produced as a byproduct of high pyruvate ferredoxin oxidoreductase (POR) activity during lactate metabolism. For these reasons, W plays a crucial role in lactate utilization in E. limosum. Low potential redox enzymes such as WOR, FDH, and POR are involved in lactate utilization and were predicted to utilize ferredoxin, a common electron carrier in anaerobic organisms. However, E. limosum has five putative electron carriers encoded in its genome, two ferredoxins (Fd1 and Fd2) and three flavodoxins (Fld, Fld-like1, and Fld-like2). Through bioinformatic analysis and biochemical characterization, Fd1, Fd2, and Fld are involved in the primary metabolism of E. limosum with a preference for Fld under lactate conditions even in iron-sufficient growth media. To gain insight into the W metabolism of a second gut microbe, Cetobacterium somerae, we determined its genome sequence that revealed two WORs, CsWOR1 (clade 41) and CsWOR2 (clade 87) as well as the tungstate transporter Tup. Based on bioinformatic analysis with the C. somerae WORs, we show that the WOR family is more diverse than we originally thought and that they can be classified into six distinct Classes depending upon subunit composition and electron carrier specificity.
