thiols and sulfur

B6 and transsulfuration

There is a connection between a sulfur salvage pathway enzyme S-adenosyl homocysteine hydrolase and vitamin B6. Both might be impacted in autism spectrum of disorders. Perhaps there is a connection in all of us. This post is what happens when one sits down with a collection of publications and asks, “How are these papers related?”

S-adenosylhomocysteine hydrolase catalyzes this reaction

According to UniProt, this enzyme binds one NAD+ per subunit. This protein is often referred to by the name of its gene AHCYL2. Let’s try to be consistent with our paper collection and call it Ado-Hcy hdydrolase. This statement comes from a 2003 study that indicates that that it’s catalytic activity is controlled by NAD+/NADH levels. [1] This group used a radioactive (tritium 3H) version of adenosine (Ado) to examine the influence of

Fig. 1. 3 H-Ado binding and enzyme activity of AdoHcy hydrolase dependent upon the NAD+/NADH ratio. Binding was performed with 10 nM 3 H-Ado and AdoHcy hydrolase 10 mg/mL. Enzyme activity was performed in the direction of hydrolysis with 50 mM AdoHcy and AdoHcy hydrolase 20 mg/mL. Data were obtained from three independent experiments.

As we shall see soon, there is a link between thiol redox status and NAD/NADH.

In a previous study cited by Koor 2003 [1] claims that the same Kloor group demonstrated that that Ado induces the reduction of the tightly bound NAD+ to NADH, resulting in enzyme inactivation. AdoHcy reconstituted with NADH can still bind with high affinity. Since Ado is removed in the reaction mixture the actual ratio of NAD+/NADH in the enzyme is unlikely to be changed during the cycle of AdoHcy hydrolysis which generates Ado. At a ratio of 90% NADH and 10% NAD+ the enzyme activity is still 30% when compared to the oxidized form suggesting that the enzyme retains sufficient activity for hydrolysis of AdoHcy at even 10% NAD+.

Twenty years later structural analyses and computational modeling have shown that using different resolved structures indicate that each monomer can exist in an “open” (bound to NAD+) or “closed” (bound to NAD+ and substrate) conformation. Upon substrate binding, an ∼18° rotation of the hinge brings together the cofactor- and substrate-binding domains, followed by a rotation of the dimers by ∼14°C

Both of these pairs are considered anti-oxidant molecules in our bodies.

Vizán ‘s phylogenetic tree has been replaced with a reminder of the connection to NAD+/NADP+. Note the nice ribbon diagram of AdoHCy hydrolase..
  • Even though a copper ion is not required for its protein stabilization or catalytic activity, Ado-HCy hydrolase is a strong Cu2+ binder both in vitro and in vivo, with a Kd of about 1 × 10–12 for free-copper, and Kd of about 1 × 10–17 in the presence of EDTA. Okay… the unanswered questions are (1) whether this enzyme can bind other heavy metals and (2) can a detoxification chelator reverse binding?
  • In vitro, copper is a non-competitive inhibitor for the substrates, facilitating the dissociation of NAD+ in a concentration-dependent manner. does any of this explain the alleged toxicity of Cu2+, especially in the liver? Does Mg2+ or Ca2+ also inhibit?
  • Although Cu-AHCY structures have not been resolved, computational predictions as well as the mechanism of inhibition suggest that Cu2+ binds the central core of the Ado-HCy hydrolase tetramer, preventing subunit interaction and/or decreasing NAD+ affinity. What about Pb2+?
  • Ado-HCy hydrolase comprises 0.5% of total hepatic cytosolic protein.

The Vizán review concluded that the interaction of cations with Ado-HCy hydrolase needs to be further investigated. [2]

A lot of the introduction seems to be using these intermediates to predict methylation status, this, that and the other. What happens during ischemia when ATP production is less? For some reason chronic renal failure was chosen because it is with elevated levels of homocysteine. [3]. The Kloor group cited references claiming that moderate hype rhomocysteinemia is commonly found in patients with cardiovascular disease while levels in chronic renal failure are considerably higher. Vascular complications are part of the pathology. Some discussion was given to the roles of intracellular and extracellular concentrations of Ado-HCy hydrolase substrates and products…

Fig. 1. Pathway of intracellular AdoHcy, AdoMet, homocysteine and adenosine metabolism. AdoHcy: S-adenosylhomocysteine; AdoMet: S-adenosylmethionine; METH: 5-methyltetrahydrofolate-homocysteine S-methyltransferase; BHMT: betaine-homocysteine S-methyltransferase; CYA:adenylate cyclase; PD1: phosphodiesterase.

Make careful note of homocysteine and cystathione beta synthase. This is where our B6 story picks up. This protein has one cofactor binding site:pyridoxal 5′-phosphate.

The chemical reaction from

Rat kidneys were rendered hypoxic by renal artery occlusion. [3]

Ischemia was performed as described in Section 4. Values are means  SEM for n animals. AdoMet/AdoHcy ratio and energy charge ,EC).P < 0:01 vs. control.a With 15 min recirculation.

This publication went on to describe potential therapeutics into the blood of these rodents.

Direct biomarkers measure B6 vitamers in plasma/serum, urine and erythrocytes, and among these plasma pyridoxal 5-phosphate (PLP) is most commonly used. This table summarizes direct and indirect ways of estimating B6 status. Let’s review these and then look and enzyme reactions in the indirect methods.

biomarkercomment 1comment 2comment 3
Plasma pyridoxal 5′-phosphatenot stablealk hos degradessensitive to glucose and inorganic phosphate
total plasma pyridoxalpros and cons yest to be evaluated
Urinary excretion of 4-pyridoxic acidimmediate response to dietPA in urine sensitive to protein intakeless sensitive to age
RBC PLPmore reliable than plasma PPno alk phos in RBCPLP binds to hemoglobin
plasma vitamer ratiospromising for some diseasesperson to person variability
CSF vitamersvariabilityin CSFsome promise
Urinary cystathionineafter oral 3g methionine↓ B6 , ↑ methionine
urinary xanthineafter oral tyrptopandata for children Sci-Hubpregnancy, hormones
Plasma kynurenineshigh renal clearancetryptophan metabolitemay require mass spec
plasma glycine↓ B6 ↑ glycine. ↑serine
A summary of the B6 status review [4]

This particular image neglected to show the oral dosing of methionine that leads to an increase in homocysteine. See above cartoon. Another metabolic test involves the role of tryptophan metabolism.

Kynurenines tend to be fluorescent. An assay has been developed this compounds in urine that is not totally clinic ready. PMC free article

Moving on to our third biomarkers, glycine and serine, things are not as straight forward.

Part of the proposal is to look at plasma glycine and/or serine. [4] All of these reactions also depend on folate and the NAD precursor niacin and on tryptophan.

Echoing the Ueland review,

  • Vitamin B6 is found in six forms: pyridoxal, pyridoxine, pyridoxamine, pyridoxic, and phosphorylated forms. [4,5] B6 vitamers participate in numerous biological metabolic pathways that include neurotransmitter synthesis and degradation that may be compromised in autism.
  • Trans sulfation pathway enzymes cystathionine beta-synthase, cystathionine gamma-lyase (in the transsulfuration pathway), and cysteine sulfinic acid decarboxylase (in taurine biosynthesis) may be particularly vulnerable in ASD.
  • Cysteine desulfurase is a B6-dependent enzyme critical for forming iron-sulfur centers like in the respiratory chain complexes I, II, and III and both mitochondrial and cytosolic aconitase. One of the products is H2S making it a bit harder to detect in the urine.
  • Another B6-dependent desulfurase pathway enzyme is molybdenum cofactor sulfurase (MOCOS) MOCOS was found to be under-expressed in some individuals with ASD due to an antisense long noncoding RNA. Here is an odd quot from UniProt that may be important, “In vitro, the C-terminal domain is able to reduce N-hydroxylated prodrugs, such as benzamidoxime.” If we are are to copy the Ueland methodolgy, we’d look for a harmless substrate of MOCOS and look for the metabolite in the urine. A in this scheme appears to be an aldehyde.
  • B6 deficiency can disrupt the immune system by changing the balance between two types of T-helper cells (Th1 vs. Th2) towards an excessive Th2 response … and so one. Disruption of the immune system may not lend itself to a B6 assay Ueland fashion.
  • Albumin bound PLP was mentioned. Humans have no vitamin B6-specific transporter. The phosphate group attached to vitamin B6 creates a barrier to its movement into or out of a cell. Inside the cell, PDXK adds a phosphate group, allowing the molecule to stay inside the cell. In words, “Catalyzes the phosphorylation of the dietary vitamin B6 vitamers pyridoxal (PL), pyridoxine (PN) and pyridoxamine (PM) to form pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate (PNP) and pyridoxamine 5′-phosphate (PMP), respectively” Mention was also made of PDXK genetic deficiency. More UniProt annotation, “Catalytic activity is inhibited competitively by 4-deoxypyridoxine, and is also inhibited by the benzodiazepine receptor ligands 1012S and ethyl-beta-carboline-3-carboxylate.
    Inhibited by ginkgotoxin, theophylline, lamotrigine, enprofylline, theobromine, and caffeine.”
  • In an additional paragraph of the Engelen autism review [5] reduced PDXK activity resulting in reduced conversion of pyridoxal and pyridoxine to phosphorylated vitamin B6 was discussed. Blood levels of PLP were found to be low in patients with bi allelic mutations in the PDXK gene. All the other vitamers of B6 tested in these patients were normal or zero instead of raised. These data are inconsistent with Adam’s hypothesis. Let’s take a look at PDXK in variant viewer. found in
Only three of the red dot variants had clinical symptoms.
  • Vanadate, oxalate, and other oxyanions are found to be ALPL inhibitors, the tissue nonspecific alkaline phosphatase ihibitor. Autistic children have higher oxalate in their urine.
  • Comments were made about RBC Band 3, aka SLC4A1, are not going to be repeated in this post.
  • Mention was made of B6 supplementation clinical trials and of potential toxicity. Pyridoxine is the standard form used in most supplements. Two studies described a paradox, showing that high concentrations of pyridoxine led to increased intracellular concentrations of pyridoxine phosphate, which competes with PLP for apoenzyme binding, decreased vitamin B6.
  • Comment was made regarding the use of B6 vitamers to accurately predict actual B6 status.
  1. Kloor D, Lüdtke A, Stoeva S, Osswald H. Adenosine binding sites at S-adenosylhomocysteine hydrolase are controlled by the NAD+/NADH ratio of the enzyme. Biochem Pharmacol. 2003 Dec 1;66(11):2117-23. PubMed Sci-Hub free
  2. Vizán P, Di Croce L, Aranda S. Functional and Pathological Roles of AHCY. Front Cell Dev Biol. 2021 Mar 31;9:654344. PMC free article
  3. Kloor D, Delabar U, Mühlbauer B, Luippold G, Osswald H. Tissue levels of S-adenosylhomocysteine in the rat kidney: effects of ischemia and homocysteine. Biochem Pharmacol. 2002 Feb 15;63(4):809-15. PubMed free on Sci-Hub
  4. Ueland PM, Ulvik A, Rios-Avila L, Midttun Ø, Gregory JF. Direct and Functional Biomarkers of Vitamin B6 Status. Annu Rev Nutr. 2015;35:33-70. PMC free article
  5. Indika NR, Frye RE, Rossignol DA, Owens SC, Senarathne UD, Grabrucker AM, Perera R, Engelen MPKJ, Deutz NEP. The Rationale for Vitamin, Mineral, and Cofactor Treatment in the Precision Medical Care of Autism Spectrum Disorder. J Pers Med. 2023 Jan 29;13(2):252. PMC free article

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