Showing papers on "Cobalamin transport published in 2007"

Structural study on ligand specificity of human vitamin B12 transporters.

Abstract: Studies comparing the binding of genuine cobalamin (vitamin B12) to that of its natural or synthetic analogues have long established increasing ligand specificity in the order haptocorrin, transcobalamin and intrinsic factor, the high-affinity binding proteins involved in cobalamin transport in mammals. In the present study, ligand specificity was investigated from a structural point of view, for which comparative models of intrinsic factor and haptocorrin are produced based on the crystal structure of the homologous transcobalamin and validated by results of published binding assays. Many interactions between cobalamin and its binding site in the interface of the two domains are conserved among the transporters. A structural comparison suggests that the determinant of specificity regarding cobalamin ligands with modified nucleotide moiety resides in the β-hairpin motif β3-turn-β4 of the smaller C-terminal domain. In haptocorrin, it provides hydrophobic contacts to the benzimidazole moiety through the apolar regions of Arg357, Trp359 and Tyr362. Together, these large side chains may compensate for the missing nucleotide upon cobinamide binding. Intrinsic factor possesses only the tryptophan residue and transcobalamin only the tyrosine residue, consistent with their low affinity for cobinamide. Relative affinity constants for other analogues are rationalized similarly by analysis of steric and electrostatic interactions with the three transporters. The structures also indicate that the C-terminal domain is the first site of cobalamin-binding since part of the β-hairpin motif is trapped between the nucleotide moiety and the N-terminal domain in the final holo-proteins.

The Disappearance of Cobalamin Absorption Testing: A Critical Diagnostic Loss

Abstract: An appreciation of the physiological peculiarities of cobalamin (vitamin B-12) is indispensable to understanding cobalamin deficiency. Almost alone among B vitamins, cobalamin deficiency is very often linked to failures of absorption (1,2). Adults rarely become symptomatic or anemic just because of poor dietary intake. One peculiarity is how tightly the complicated, high-affinity binding proteinand receptor-dependent absorptive process built around gastric intrinsic factor (IF), and around other binding proteins throughout the body, controls every aspect of cobalamin transport and transfer (2). The IF system assures both delivery from limited animal-derived dietary sources and reabsorption of biliary cobalamin, partially explaining the often muted clinical impact of poor intake. Yet the saturable system’s own somewhat limited capacity and subtly restrictive, even exclusionary features (perhaps meant to exclude nonfunctional cobalamin analogs) also create inefficiencies that are not always obvious. Barely .1 mg of cobalamin is made available from most meals no matter their cobalamin content, and then only if the IF-mediated process functions properly (2,3). The greatest weakness, however, also lies in the heavy dependence of absorption, and thus cobalamin status, on the IFbased system, whose sole back-up, a nonsaturable but dismally inefficient carrier-free diffusion, can transport only 1% of presented cobalamin. Should IF secretion be lost (‘‘pernicious anemia’’) or its delivery of cobalamin to IF receptors fail (intestinal malabsorption), it cannot be compensated dietarily except with cobalamin intake so massive as to be unavailable in nature. As much as 1000 mg would need to be ingested daily to compensate for the failure of both absorption and enterohepatic reabsorption by the IF system, but very few food items contain .1–2 mg/ 100 gm portion (3). The carrier-mediated and carrier-free absorption processes of most other B vitamins, such as folate, are more effective. An important saving grace derives from another peculiarity of cobalamin, its slow turnover. Except for rare defects of cellular utilization, whatever causes cobalamin deficiency in adults must persist for several years to deplete body stores (which exceed daily intake 1000-fold) to the point of clinical consequences. Many potential disturbances, whether malabsorptive or not, are too transitory to produce clinical cobalamin deficiency or often even subclinical deficiency, which consists of biochemical changes without apparent clinical consequences (2,4). Absorption testing Because a clinical diagnosis of cobalamin deficiency, with hematologic or neurologic changes, implies the presence of long-standing gastrointestinal disease until proven otherwise (1), absorption testing has always been an essential task for clinicians and investigators. The traditional Schilling test, designed in 1953, identifies malabsorption by the poor urinary excretion of an orally administered cobalamin dose. When repeated along with a dose of IF, the test also distinguishes between gastric and intestinal defects. If malabsorptive processes stay undiagnosed, management decisions and vitamin replacement strategies tend to be haphazard and incomplete (Table 1). In 1973, Doscherholmen and Swaim (5) expanded the scope of malabsorption to include food-cobalamin malabsorption (FCM), an inability to release food-bound cobalamin and make it available to gastric IF. FCM cannot be diagnosed with the Schilling test, whose radiolabeled test dose of free cobalamin bypasses the need to release food-bound cobalamin. Work from many laboratories in the 1980s and early 1990s established that FCM was associated with 30–50% of all low cobalamin levels, a frequency at least 10-fold that of the more clinically ominous malabsorption of free cobalamin that occurs when gastric IF secretion or its uptake by intestinal IF receptors fails (6). However, the individual impact of FCM on cobalamin status is typically milder and more delayed. That is because FCM affects only a preparatory step and thus compromises rather than abrogates the final IF-mediated steps of absorption (and presumably does not impair reabsorption of biliary cobalamin). Pernicious anemia, the absence of IF, was originally lethal and even now carries the risk of neurologic deterioration if not properly diagnosed and treated (2,3), whereas most persons with FCM have asymptomatic, subclinical cobalamin deficiency or sometimes no deficiency at all (6,7). Numerous studies explored absorption testing methods and the mechanisms of FCM, as reviewed elsewhere (6). It became evident that the mechanisms were more diverse than initially suspected, that FCM does not always require atrophic gastritis and achlorhydria, and that our understanding of FCM and its implications was incomplete (5–7). As one poorly understood example, the intriguing reversal of FCM after antibiotic treatment has been attributed separately to Helicobacter pylori and facultative anaerobes, although neither organism’s role was identified or directly proven (4,7–10). FCM testing never became clinically available and much work remains to be done (6,7). 1 Author disclosures: R. Carmel, no conflicts of interest. * To whom correspondence should be addressed. E-mail: rac9001@nyp.org.

Crystallization studies of 5'-deoxyadenosyl radical enzymes

Abstract: ion from a cysteine thiol, which then catalyzes the reduction of ribonucleotides (Figure 1.3). While the overall mechanism of this reaction varies greatly from that of the isomerases, in every case, the reaction involves abstraction of a hydrogen atom by Ado*. 1.2.3. Adenosylcobalamin transport and activation For AdoCbl-dependent isomerases in the absence of substrate, the cobalamin homolysis products are not observable in solution; however, upon substrate binding, the homolytic cleavage rate is increased a trillionfold (4). An important question in the study of AdoCbl enzymes is how the enzyme avoids generating potentially harmful radicals in the absence of substrate, and how the substrate binding increases the cleavage rate so dramatically. In addition, the organism must avoid generating radicals from AdoCbl before it is loaded on the enzyme. This is important both in protecting the cell from radical damage and protecting the cofactor from destruction, since cobalamin biosynthesis requires a lot of energy. The cobalamin transport system in humans has been studied extensively, and it appears as though cobalamin is essentially always protein-bound in the body (5). While many different AdoCbl isomerases are found in bacteria, humans use AdoCbl in only one enzyme, MCM, which performs a carbon skeleton rearrangement to convert methylmalonyl-CoA into succinyl-CoA. If this enzyme is inactive, methylmalonic aciduria, a potentially fatal condition, can result. It has been suggested that human adenosyltransferase (hATR) both catalyzes the reaction that forms AdoCbl from cobalamin and adenosine 5'-triphosphate (ATP) and acts as a chaperone, handing off AdoCbl to MCM (6). This delivery service would prevent side reactions that could occur if the AdoCbl was floating around in the cell. Once the AdoCbl is in place on the enzyme, it is also necessary to control reactivity to prevent radical damage to the enzyme. In the case of MCM, this is achieved in part through the coupling of substrate binding to C-Co bond homolysis (7), and in part through protection of the enzyme by MeaB, a chaperone that binds to MCM and prevents inactivation (8). 1.3. Adenosylmethionine radical enzymes AdoMet has long been known as a methylating agent used in many pathways in the cell (reviewed in 9). More recently, enzymes that use AdoMet as a free radical initiator have been characterized (reviewed in 1). AdoMet radical enzymes participate in biosynthetic and catabolic pathways and are present in all three kingdoms of life. All AdoMet radical enzymes contain a CxxxCxxC motif, with the three cysteines coordinating three of the irons in the [4Fe-4S] cluster (10). The fourth iron of the [4Fe-4S] cluster is coordinated by AdoMet itselt by its amino group and carboxylate oxygen (11). 1.3.1. General mechanism The cluster must be reduced from its resting state of [4Fe-4S] 2+ to [4Fe-4S]'+ for activity; in E. coli, this reduction is catalyzed by flavodoxin (12). The reduced cluster transfers an electron to AdoMet, reductively cleaving the carbon-sulfur bond to produce methionine and Ado*, while simultaneously regenerating the [4Fe-4S]2+ cluster (Figure 1.4). The carbon-sulfur bond cannot be cleaved homolytically as in AdoCbl-dependent enzymes because the bond is too strong (greater than 60 kcal/mol) (13). After cleavage, the 5'deoxyadenoxyl radical abstracts a hydrogen atom from the substrate. After the reaction occurs, in some cases (lysine 2,3-aminomutase (2,3-LAM), spore photoproduct lyase), the AdoMet is re-formed (1); in other cases (lipoate synthase (LipA), class III RNR, Biotin Synthase (BioB)), methionine and 5 '-deoxyadenosine are products of the reaction (1). 1.3.2. Reactions catalyzed by AdoMet radical enzymes AdoMet radical enzymes catalyze reactions on a huge variety of substrates, ranging from as small as the single amino acid lysine to large proteins (Figure 1.5). These reactions include amino group migration, carbon-carbon bond cleavage, carbon-sulfur bond formation, alcohol oxidation, and glycyl radical formation. While the substrates vary greatly in size and the reactions vary greatly in outcome, all are initiated by the abstraction of a hydrogen atom. In the class III RNR activating enzyme and pyruvate formate lyase activating enzyme, as well as other enzymes, the AdoMet radical enzyme abstracts a hydrogen atom from a glycine residue in another protein; this glycyl radical then goes on to catalyze another reaction. In the case of class III RNR, this glycyl radical abstracts a hydrogen atom from a cysteine, forming the thiyl radical necessary for ribonucleotide reduction with a similar mechanism to the other classes of RNR. 1.4. Adenosyl radical chemistry and enzymology AdoMet radical enzymes use a [4Fe-4S] cluster and AdoMet to create Ado*, while adenosylcobalamin (AdoCbl)-dependent isomerases also generate Ado* using coenzyme B12. Both AdoMet radical enzymes and AdoCbl isomerases can have TIM barrel folds or partial TIM barrel folds where the radical chemistry occurs (14). TIM barrels were first discovered in the enzyme triose phosphate isomerase and are ubiquitous in nature. They are most often involved in energy metabolism, but are present in at least 28 different enzyme classes. A full TIM barrel consists of an (oW)8 motif. The #-sheets line the interior of the barrel, which can protect reactive intermediates from solvent. While in the solved structures of AdoCbl enzymes, the TIM barrel is the full (4o)8 barrel (15, 16, 17, 18, 19, 20), three out of the four structures of AdoMet radical enzymes have a threequarters (01)6 barrel (21, 22, 23, 24). Superposition of the TIM barrel of AdoCbldependent diol dehydratase with that of the AdoMet radical enzyme BioB shows that the ring of diol dehydratase's AdoCbl occupies the same position as the [4Fe-4S] cluster in