The adventures of Folate coenzymes and folate: from bacteria playing dead to fortified bread,  hopefully one day coming to a theater near you! Even superheroes need sidekicks – you might remember the bumbling biochemist’s trusty sidekick Steppy the Stepstool… Well, even some enzymes need sidekicks – small molecules called coenzymes that bind to the enzyme (which is usually a protein, sometimes a protein/RNA combo, or even just RNA alone) and help the enzyme carry out its task of speeding up (catalyzing) biochemical reactions. By doing things like holding interacting partners together in the optimal orientations and chemical environment, enzymes are able to speed up processes like molecule-making (e.g. DNA writing) and molecule-breaking (e.g. DNA cleavage) by quadrillions of times (and I’m not exaggerating here!).

But that can be a hard task – for simplicity let’s just consider protein enzymes. Proteins are made up of chains of amino acid “letters” – there are 20 (common) amino acids and they have different properties, from small & flexible to big & bulky, water-loving (hydrophilic) to water-excluded (hydrophobic), negative to neutral to positive. Those properties influence how the protein folds so it can help the protein adopt shapes that are great for binding different things. But its willingness to bind things is limited by that one set of amino acid letters. And there are countless possible molecules that could serve as reactants. And enzymes have to be super-specialized to only bind and help convert super specific ones.

We use the term “substrates” for the reactants an enzyme binds and changes into product. Might seem like a silly semantics thing, but using the term “substrate” (a thing an enzyme binds and helps convert to product) instead of “ligand” (a thing that something binds) is a great reminder that an enzyme not only has to bind specific things, but it also has to be able to help change, and that change could be all sorts of different things (breaking up, adding on, etc. – though each enzyme is specialized to just do one thing (typically)). And, to further challenge things, enzymes, by definition are catalysts, meaning that they don’t get used up in the process of helping out – so they can do it over and over and over. 

This substrate-selection-and-selective-changing-super-specificity often calls for expanding the available options past what is available through amino acids alone. And this is where coenzymes, like folate, can come in. 

I’ve known for a long time that folate (or folic acid as it’s commonly added) is added to bread and other bread-y things – you can’t go far without seeing something like “folate-fortified” on a label. And I knew it had something to do with pregnant women, but that was about it. But then I was reading this book by Arthur Kornberg which IUBMB president-elect Dr. Alexandra Newton gave me, “For the Love of Enzymes” – which I love by the way & highly recommend. And in it, Kornberg tells the cool study of the discovery of folic acid. Turns out it’s tied to the discovery of “sulfa drugs” (early antibiotics), and, in the early 1920s, a doctor named Lucy Wills discovered that she could reverse or prevent a blood disorder called prenatal macrocytic anemia by supplementing the women’s diets with a yeasty spread called Marmite. It was later shown that the key molecule in the Marmite, the “Wills factor” was folate. Folic acid supplementation has since become standard for pregnant women, and saved the lives of countless mothers and children.

In the early days of science, people called vitalists thought that there was this kinda essential essence of life that made possible all the complex reactions and interconversions of molecules that let the cells in organisms do their stuff. But then scientists started showing that you could break open cells and still get stuff to happen – and you could even purify out various components and then mix those components to get stuff to happen. And there were proteins called enzymes that could make those reactions happen much faster (they didn’t know at the time what these speed-uppers actually were, so they called them ferments because if you added them to grape juice they could get it to ferment into wine.

Scientists then started racing to discover what the minimal requirements for life were – they fed animals (and later bacteria which are much easier to work with) strict diets and looked to see what happened when those diets were lacking certain things. They often didn’t initially know what the “lacking things” were and often went looking for the wrong type of thing because they weren’t expecting “boring little molecules” to have such an impact – they’d only just gotten used to the idea of bacteria causing diseases and stuff!

For example, take the case of a disease called beriberi (characterized by symptoms including weakness, paralysis, liver disease, and heart failure). In the late 1800s, Japanese sailors were eating “polished rice” which was rice which had the husks removed. It was considered more of a “luxury item” but it actually ended up leading to the death of a lot of sailors. A ship doctor named K. Takaki put the link together between diet and beriberi – he did a controlled experiment where he fed some of his crew polished rice (the traditional Japanese sailor’s fare) and the rest of the crew the British navy’s standard fare (oatmeal, veggies, fish, meat & condensed milk) – 2/3 of the rice-fed sailors got beriberi but NONE of the British diet. He didn’t know why this was but, by changing to a new diet, thousands of beriberi deaths were prevented. And it wasn’t just that British diet in particular – natural “husked” rice could suffice – so some scientists thought that there was some toxin in the rice that the husk had an “anti-toxin” for. 

Some of those early vitamin-hunting stories were from “natural experiments” – scientists trying to figure out what was going on in people or animals “in the wild.” But they later turned to more controlled settings. Arthur Kornberg would later win the Nobel Prize for his work on DNA-making (he identified and isolated DNA Polymerase, an enzyme which can copy DNA by using a template strand to lay down the complementary strand), but one of his earliest research projects was one of those “What’s missing” experiments. Only this time, instead of “What’s missing?”  it was more like “What’s being stolen?”

He had a bunch of rats and they were fed a defined, minimal diet composed of purified known ingredients with precise portion sizes – it had everything the rats needed to survive, but it wasn’t 5-star cuisine or anything – the rats got the main macromolecules (carbs, proteins, and fats) through sugar in the form of glucose (73% of their diet), protein in the form of vitamin-free casein (milk protein) (18%), fats in the form or cod liver (2%) and cottonseed (3%) oils. And they got other, smaller but still super important molecules through supplements of salts (4%) and the vitamins thiamine, riboflavin, pyridoxine, niacin, & pantothenate (and choline).

It might not have made the rodent Zagat guide, but this diet was enough to allow the rats to grow & thrive – it had everything they needed – or did it? Turns out they weren’t taking into account what the organisms inside the rat needed! 

Bacteria are often seen as “enemies” and there are some harmful ones, but there are also lots of good ones, and we have a diverse lot of those good guys living in our digestive tract (you’ve likely heard of this “microbiome”). The bacteria like to live there because the food we eat provides them with nutrients they need. But they don’t just steal our nutrients – they turn “pre-nutrients” into versions of nutrients that our cells can use.

Often they can make (synthesize) molecules that we need but which our cells don’t have the machinery to make. The machinery needed is enzymes fit for the task. Enzymes are really specific so you need different enzymes to carry out the different steps of making different molecules and, since bacteria need to make things that we don’t have to – like cell walls (our cells are only surrounded by lipid membranes, not carb-y walls)) – bacteria need to have enzymes that we don’t have. 

So these bacteria-specific enzymes make attractive targets for antibiotics – we can target them without having to worry about accidentally targeting enzymes in our own cells. There are different types of enzyme inhibitors – some (the competitive inhibitors) mimic the real substrate and compete with  it for the binding site, whereas others (uncompetitive & noncompetitive inhibitors) bind elsewhere on the enzyme & keep it from working. 

A lot of enzyme inhibitors are reversible – they bind to the enzyme but can be competed out by the real thing in the case of competitive inhibitors or diluted out in the case of the others – so the inhibition’s temporary. But some enzyme inhibitors are what we call “irreversible” – they bind in such a way that there’s no going back, so the enzyme’s permanently out of commission. 

An example of this type of inhibitor is the antibiotic penicillin – it works by inhibiting a bacterial enzyme called transpeptidase, which the bacteria need to reinforce their cell walls to keep their cells from bursting like a water balloon. Ampicillin works by mimicking the enzyme’s normal substrate, tricking the enzyme into binding it. And the enzyme tries to use it to build its wall. But the Amp may be similar enough to bind, but it’s not similar enough to wall-build with, so the enzyme gets stuck. And to make things worse for the bacteria, this sticking is permanent because Amp forms a strong covalent bond with the enzyme that permanently prevents the enzyme from working 

Penicillin is rightly hailed as ground-breaking discovery that saved (and continues to save) countless lives. But penicillin is far from the only antibiotic – and the 1st class of antibiotics discovered were the so called “sulfa drugs” (aka sulfonamides) First used in the 1930s, as they came into widespread use, scientists wanted to be super sure they wouldn’t cause more harm than good. So they gave sulfa drugs to some healthy rats – and to avoid differences in diets from confuddling their findings those rats were given that defined, minimalist diet. And what they found surprised them – these “miracle drugs” were causing the once healthy rats to develop blood disorders and die. 

The rats developed 2 main types of blood disease – one you’ve probably heard of, the other likely not. The first was anemia – anemia is actually a pretty broad term that covers a wide range of conditions that lead to decreased red blood cells – which is a serious problem since these RBCs are needed to carry oxygen throughout the blood. The second type of blood disorder they saw was granulocytopenia – granulocytes are a type of white blood cell, important for the immune system, and they were dying off too.

The scientists set out to find what could reverse the effects – and they found that if they supplemented the sulfa-treated rats’ diet with a liver supplement they could cure the mice. And if they pre-supplemented the rats before giving the sulfa drugs the rats avoided the blood problems all together. Kornberg’s rats weren’t the only ones experiencing such problems – other groups found similar results (healthy animals sickened by sulfa drugs) with monkeys and chickens – and they found there was some thing in liver (and yeast, and vegetables) that could prevent or cure the anemia.

Why could this be?

Targeting bacteria-specific enzymes (molecular machines that bacteria have & need but our cells don’t) keeps our cells from being harmed, but if the “good bacteria” are prevented from making something we need from them, we can be “indirectly harmed.” If sulfa drugs were causing anemia because they were killing off the good bacteria that were making something the animals needed to keep their blood cells happy, scientists wanted to find what the “something” being made was. And to do this, they searched for bacteria that couldn’t make that something.

It’s really hard to look for something when you don’t know what you’re looking for – and it’s not even like bacteria were the ones developing anemia! So they had to find bacteria that couldn’t grow without the same thing that could cure those rats. And then they had to figure out what that thing was. 

They searched for bacteria that couldn’t grow without those yeast, liver, etc. extracts that the sulfa-fed rats needed. It might sound that a monumental undertaking, but since bacteria divide so rapidly and can be kinda sloppy with their genetic proofreading, it’s easy for mutations to occur – and if those mutations occur in one of the enzymes needed for making a molecule, it can prevent that molecule from being made. So bacteria with those mutations can’t grow unless that molecule is given to them pre-made in their food 

Some “natural” or “wild strains” of bacterial species are missing key enzymes and other times mutants arise after lots of lab growing. The folic acid work was done on some of those “wild strains”

E.L.R. Stokstad at Lederle Laboratories in New Jersey was working with Lactobacillus casei (L. casei) which is important for cheese fermentations. He found it couldn’t grow in culture medium that didn’t have liver or yeast extracts – but he didn’t know what it was in those extracts that the bacteria needed but couldn’t make. So instead of adding whole extracts he purified out various molecules from the extracts and added them one at a time to see which allowed the bacteria to grow. 

The same molecule was identified by similar work carried out by Herschel Mitchell in 1941 with a different species of bacteria, Streptococcus lacti. Instead of using liver or yeast, he gave his bacteria a different source of feast – he purified a substance from spinach – a lot of spinach – 2 tons of it gave him 10mg. It had acidic properties and it came from a leafy thing, so he named it folic acid (folium is Latin for leaf). 10mg might not seem like much, but only 25 micrograms (so 1/400th of that spinach yield) a day could cure mice with that fatal low white blood cell problem in just 4 days. 

And something potentially related was happening in humans too – an English doctor named Lucy Wills found that supplementing the diet of pregnant women with yeast or liver extracts prevented them from devoting a severe anemia that was commonly seen. (more on her later)

In 1943, Stokstad crystalized the miraculous mystery molecule and figured out its structure. What he found the structure of was the simplest folate – and its finding was probably an artifact of long isolation procedures in which natural folate decomposed to their simplest form – folic acid (pteroylglutamic acid). “Folate” includes folic acid and the natural form(S) – plural – there are lots of different forms of it – potentially as many as 150, but probably more like 50 or so – all based around this same general structure, but with different modifications.

Folates have 3 main parts

  1. on one end is a “pteridine ring” – it gets that name because it’s part of a group of chemicals with a similar shape that are found in the pigments that give butterfly wings their color (and butterflies are lepidopterans) 
  2. in the middle is a PABA group – PABA stands for ParaAminoBenzoic Acid
  3. at the other end is glutamic acid (one of the protein letters (amino acids)

Put those 3 things together and you get the mouthful of a name pteroylgutamic acid (PGA). Folic acid is the fully oxidized version – it’s basically the simplest version. It’s not this simple, but you can kind of think of the oxidized sites as being places where things can be attached – oxidation refers to the loss of electrons – it’s opposite is reduction – and when you add electrons you can also add other things with them.

common modifications found in natural folates are  

  1. addition of more glutamates (poly glutamates) (often 5-8)
  2. reduction to tetra-hydroforms
  3. single carbon containing (1C) units (things like methyl (-CH₃), formyl (-CHO), methylene (=CH₂) & methenyl (=CH₄) linked up to the 5th and/or 10th nitrogen atoms. 

Those 1C units are really important because it allows folate to act as a single-carbon carrier.  It often gets its 1C units from donors like the amino acid (protein letter) serine. And then it can transfer those molecular building blocks to other molecules to make bigger molecules. This building block transferring is sped up by enzymes – what usually happens is that enzymes hold onto folate and use them as helpers – we call such a combo a “folate coenzyme” and there are a variety of them involved in making different things. 

One such molecule it’s needed for making is DNA – folate coenzymes are required for making DNA letters from their thymidine & purine precursors.And it’s also needed for making some protein letters (amino acids) – methionine, cysteine, serine, glycine, and histidine – in addition to its role in protein-making, methionine (Met) can then be used as a 1C donor for making another 1C donor (S-adenosylmethionine (SAM) which methylates DNA and stuff to help regulate gene expression. 

Pregnant women have a larger requirement for folate because they have to make molecules for not just themselves but for their babies too. Neural tube defects (NTDs) including anencephaly &  spina bifida are problems where neural tubes (embryonic precursors to the nervous system) don’t fully close when an embryo is forming, leading to problems including paralysis. Neural tube closure happens really early in pregnancy (about a month after conception) so by the time women know they’re pregnant, it’s usually too late – so countries started putting into place mandatory fortification requirements – for example, in 1998 the US mandated that enriched cereal grain products be fortified with 140ug (micrograms) of folic acid per 100g), This led to a decrease in NTDs of 19-32%.

And for this we have to thank hematologist Dr. Lucy Wills. Wills was born in England in 1888. She attended a British boarding school called Cheltenham College for Young Ladies (one of the first such schools to teach girls science and math) and In 1911 she graduated from Cambridge University with degrees in geology and botany. She then received medical training at the London School of Medicine for Women.

She decided to go into research and, in the 1920s and 1930s she traveled to India to investigate a public health crisis; pregnant textile workers in Bombay were developing a condition wherein their red blood cells were becoming abnormally large and “diluting” the blood’s ability to transport oxygen. She searched extensively for a bacterial culprit, but couldn’t find one. But she did find one connection – it seemed that only poor women were becoming ill. She wondered if a dietary factor could be involved, so she carefully recorded their diets.

She fed a group of monkeys the same diet and one developed similar symptoms – but if she supplemented the monkey’s diet with a yeast-based spread called Marmite, the monkey got better. She didn’t know at the time what it was in the Marmite that saved the monkey (or the rats she tested) but she had identified a dietary connection and found a cheap cure and prevention strategy.  It was later shown that the key molecule in the Marmite, the “Wills factor” was folate. Folic acid supplementation has since become standard for pregnant women, and saved the lives of countless mothers and children.

This fortification is done with folic acid instead of a natural folate because it’s more stable (think back to why Stokstad found it in this form) and readily absorbable. And even though it’s not the form our cells use directly, our bodies can convert it to usable forms. Our body has to convert natural folate from our food before we can take it into our cells anyway. Folate in the food you eat gets simplified before your intestinal cells can take it in – the polyglutamates are turned into monoglutamates (all but one are “chopped off” through hydrolysis (bond cleavage aided by water)). Then they’re taken in & a methyl (-CH₃) group is added to the 5th nitrogen to give you 5-methyltetrahydrofolate (5-MTHF) the major circulating form in humans. 

What does this have to do with sulfa drugs – those antibiotics causing anemia in mice? Sulfa drugs look really similar to that PABA group (the middle part of folate) so they can confuse folate-making enzymes. The reason the sulfa drugs affect bacterial cells but not our cells is that our cells couldn’t make folic acid to begin with – bacteria can make it (and most have to make it because it can’t pass through their cell walls unless they have special helpers). Remember how folate as those 3 parts (pteridine, PABA, & glutamate)?. Well, they make it by first combining PABA with dihydropteroate diphosphate (the pteridine part) with the help of an enzyme called dihydropteroate synthase to form dihydropteroic acid and then linking that up to glutamate with the help of dihydrofolate synthase to get a folate. 

What was happening was that the bacteria would go to make folate, but in the first step they’d grab the sulfa drug instead – and they’d get tricked into using it – so they’d waste a pteridine on the faker. And then the second enzyme – the dihydrofolate synthase – well, she’s not fooled. So she wouldn’t add the glutamate, so less folate would be formed and bacterial growth would slow – but unlike with the bactericidal ampicillin, which kills the bacteria outright, the sulfa drugs are bacteriostatic – they slow growth but don’t kill the cells – at least initially. Also unlike the ampicillin, these sulfa drugs are reversible, competitive inhibitors – they compete with the real substrate, but the real substrate can outcompete it – in fact, scientists could inject just the PABA part and it would prevent the sulfa drugs from causing anemia. 

This post is part of my weekly “broadcasts from the bench” for The International Union of Biochemistry and Molecular Biology. Be sure to follow the IUBMB if you’re interested in biochemistry! They’re a really great international organization for biochemistry.

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉

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