Hi James, you can do a BLAST of your enzyme to see if homologous dioxygenases require cofactors.  

If you enzyme requires, for example, a heme cofactor then it will not be present in the bugs no matter the media.

LB media is a rich media and should indeed normally give enough cofactors to give at least some small amount of activity. However, media change from batch to batch and from manufacturer to manufacturer. Sometimes wildly. Re: heme in particular, heme dioxygenases are relatively rare and I work on IDO, which is one. Ecoli certainly can make a reasonable amount of heme for incorporation. This can be increased by using its biological precursor, ALA. Hemin can also be used to (usually) lesser effect (and can sometimes be toxic). I also make an oxidoreductase that requires both fmn and fad. Again, on small scale expressions, both are there from Ecoli growths and the enzyme is active.

Re: finding the specific co-factor for yours, using BLAST to find homology is indeed the best starting point. Followed by using those cofactors both during growth and during assasys (as initial tests of where best to use it). Ecoli can be bad at making riboflavin based co-factors due to a lack of active transport mechanisms. KMO can benefit during assays by the addition of fad as it is not co-valently linked and can move in and out of the protein.

Ofcourse, there is no guarentee that you protein is even expressed full-length or folding correctly in the first instance, and therefore it might have nothing to do with co-factors or His tags.

Is it the correct size on sds-page and MS? What does size exclusion FPLC look like? Was it a bacterial or animal lysate used before? Could it require another protein partner to operate?

I haven't noticed any issues with expression or purification.  The protein is correct size according to SDS-PAGE and is visibly more intense after induction using IPTG.  The confusing part is that the recombinant enzyme was previously shown to be active using E.coli cell lysate (the enzyme was discovered in strain of Rhizobium originally, now it is expressed using E.coli).  According to previous work, over the course of four hours, 0.1mM of the substrate was fully degraded by cell lysate of 1 mg/mL total protein concentration.

Since I also tested the lysate, I didn't think that there would be another protein partner involved because the enzyme was not active after purification or in the cell lysate.  The only difference between LB and YEM media that may be affecting the protein function is Ca and Mg concentration (much higher in YEM).

Growing the E.coli cultures as the literature described, by supplementing the LB media with iron, I assumed that iron may have had a role in correct folding as well as activity.

 Oh, well...tomorrow I'm going to throw every metal cofactor in to tool box at it and if all fails.....try sequencing the insert DNA then?

If there is any doubt as to the plasmid, then yes I would certainly target sequencing as well as making sure your antibiotics are actually still active. I once had a cell line throw a plasmid because my ampicillin wasn't active - I now run MIC assays every 12 months. Do you use protease inhibitors? I use PMSF during growth and then edta-free cocktail inhibitors during purification to prevent deactivation of the enzyme by cleavage of a small peptide.

https://www.bnl.gov/biosciences/staff/Studier/files/StudierRecipes07-12-20.pdf

This resource has a number of Studier's recipes for BL21 growths, including a trace metals recipe. 

If all else fails, there is also the possibility of considering the original assay for the previous lysate. Were controls with empty vector used to determine what the background metabolism rate was? Is the assay specific for the substrate? Under what conditions could it give false positives or false negatives? Is the substrate still the substrate or could it have degraded during storage? Was the lysate always collected at the same cell-cycle stage? Does the lysate of a non-induced culture support the reaction?

Just read your inquiry and the immediate responses are similar to Jason's responses:  1) correct mass spec'd-protein that's not altered from folding, degradation, or sequence issues,  2)  is the assay validated, 3) LB-grown cell pellet was broken in YEM and lysate tested,  If iron is the concern then during growth, while the protein is being made/folded with iron, there may not be sufficient  bio-available Fe in which case siderophores are produced by E coli and there's not much in the world that can compete with siderophores for iron.  Siderophore-Fe is usually a pink/red complex.  

Most extradiol dioxygenases require ferrous iron. I would suggest supplemnting the media with ferrous ammonium sulfate and even adding it to purification buffers. There is also one known extradiol dioxygenase that requires manganese.

R u looking for a coenzyme or a cofactor? How about denaturing, either by heat or TCA , any active enzyme prep and use their supetnatants, properly neutralized for the latter case, as additives in UR inactive preparation. If the activity is restored to 30-40% ofvur expectation, then its easy to try minerals/coenzymes. If not, then UR recombinant may have folding problems. Note, His tag will use Select metal for binding. 

Do you have purified active enzyme from Rhizobium that can be subjected to ICAP (inductively coupled argon plasma) analysis for metals?  It's extremely sensitive and covers a wide range of elements.  Extraction of a complete protease digestion may also release any bound solutes then try mass spec of the digest extract?

I don't have the purified enzyme from Rhizobium....I may be able to dig up the particular strain of Rhizobium in the freezer but that wouldn't help.  It is possible for me to do ICP-MS but I doubt that would help either because my enzyme is not active.

Is it unlikely that the cell culture and pure protein requires a metal cofactor to be present all through cell growth, translation, and purification?  I assume that it is sufficient to provide the cofactor in the assay mixture...I've been using a small amount of Ferrous sulfate (100uM) and aspartic acid because the substrate chelates iron.

I think ferrous sulfate is doing the trick, still working on confirming that though...