Improving Codeine conversion via enzyme manipulation

[serotonin2A]

How would one be able to find the correct aa sequence for the new enzyme so that it catalyses the proposed reaction well? It seems like an incredibly hard task. Has it ever been done before with other enzymes?

There are a few strategies that biochemists used to engineer new enzymes. You can try to make chimeras of other enzymes. You can also make many many progressive rounds of directed or random mutations, stopping at each step to see if activity improves. If you know the 3D structure then you can play around with the model and try to figure out where the catalytic residues need to be positioned.

 

[aced126]

There are a few strategies that biochemists used to engineer new enzymes. You can try to make chimeras of other enzymes. You can also make many many progressive rounds of directed or random mutations, stopping at each step to see if activity improves. If you know the 3D structure then you can play around with the model and try to figure out where the catalytic residues need to be positioned.

Seems like a lot of trial and error. If the positions of the new catalytic residues are found, then how does one know the protein will even fold up into the right conformation when made?

 

[serotonin2A]

Seems like a lot of trial and error. If the positions of the new catalytic residues are found, then how does one know the protein will even fold up into the right conformation when made?

You won't know until you make the enzyme and measure the activity.

Here's an example of one approach:
http://www.nature.com/nchembio/journal/v9/n8/full/nchembio.1276.html

 

[PINKoPANaTHER]

^ This proposed idea could take several years and millions of dollars to do, although I have read somewhere (I think popular science) that they already have created either an S. Cerevisiae or E. Coli strain with the proper plasmids/genetic alterations to produce morphine, but the cost, harvest, and total production is absolutely untenable compared to simply growing poppies and synthesizing compounds from the alkaloids.

I've created probably 30-40 spliced, truncated, point mutated, or otherwise altered genes before and each one can take weeks, maybe even months at a time, especially in your standard academic lab. You go in with a basic hypothesis that amounts to a shot in the dark, even with years of clear crystal structures and binding/activity data. Whereas the cost of growing massive amounts of poppies and synthesizing hydromorphone is easy, and actually pretty damn cheap. I also believe there is a drug (recerational) that when mixed with hydrocodone induces a significantly larger proportion of it to be converted to hydromorphone.

An interesting idea, of course, but practically unfeasible.

 

[clubcard]

I read a long time ago that 2D6 is particularly difficult to find inducers for (and research went into this), glutethimide being one of the known inducers. I remember reading a 1 year diary of Nikki Sixx who said that when his dealer had them, he would bring them over (Sixx was doing so much crack & H, this was 'on the house'). It was '3 of these weird painkillers and a couple of weird sleeping pills' and 'you were flying higher than a speedball' Now, the gentleman's memory was hazy due to his massive drug intake but for HIM to be bowled over by them, the effects must have been.... potent. Codeine & dihydrocodeine are all sold [P] and I was going to explain how to remove the unwanted... then I realized I would get thrown off the site.

I hope it's OK to say -

1- check solubility
2- check solubility of salts

In no way am I in favour of opiates. In the circles I moved in when younger, I saw to many people die, to many people with hepatitis and too many people starting with a DVT which moved, and killed them. Opiates can make a useful servant - but it's a terrible master.