Jessica Momb

Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice

Maternal supplementation with folic acid is known to reduce the incidence of neural tube defects (NTDs) by as much as 70%. Despite the strong clinical link between folate and NTDs, the biochemical mechanisms through which folic acid acts during neural tube development remain undefined. The Mthfd1l gene encodes a mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase, termed MTHFD1L. This gene is expressed in adults and at all stages of mammalian embryogenesis with localized regions of higher expression along the neural tube, developing brain, craniofacial structures, limb buds, and tail bud. In both embryos and adults, MTHFD1L catalyzes the last step in the flow of one-carbon units from mitochondria to cytoplasm, producing formate from 10-formyl-THF. To investigate the role of mitochondrial formate production during embryonic development, we have analyzed Mthfd1l knockout mice. All embryos lacking Mthfd1l exhibit aberrant neural tube closure including craniorachischisis and exencephaly and/or a wavy neural tube. This fully penetrant folate-pathway mouse model does not require feeding a folate-deficient diet to cause this phenotype. Maternal supplementation with sodium formate decreases the incidence of NTDs and partially rescues the growth defect in embryos lacking Mthfd1l. These results reveal the critical role of mitochondrially derived formate in mammalian development, providing a mechanistic link between folic acid and NTDs. In light of previous studies linking a common splice variant in the human MTHFD1L gene with increased risk for NTDs, this mouse model provides a powerful system to help elucidate the specific metabolic mechanisms that underlie folate-associated birth defects, including NTDs.

Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 1. Product-Bound Structures†‡

Enzymes capable of hydrolyzing N-acyl-l-homoserine lactones (AHLs) used in some bacterial quorum-sensing pathways are of considerable interest for their ability to block undesirable phenotypes. Most known AHL hydrolases that catalyze ring opening (AHL lactonases) are members of the metallo-β-lactamase enzyme superfamily and rely on a dinuclear zinc site for catalysis and stability. Here we report the three-dimensional structures of three product complexes formed with the AHL lactonase from Bacillus thuringiensis. Structures of the lactonase bound with two different concentrations of the ring-opened product of N-hexanoyl-l-homoserine lactone are determined at 0.95 and 1.4 Å resolution and exhibit different product configurations. A structure of the ring-opened product of the non-natural N-hexanoyl-l-homocysteine thiolactone at 1.3 Å resolution is also determined. On the basis of these product-bound structures, a substrate-binding model is presented that differs from previous proposals. Additionally, the proximity of the product to active-site residues and observed changes in protein conformation and metal coordination provide insight into the catalytic mechanism of this quorum-quenching metalloenzyme.

Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate Modeling and Active Site Mutations

The N-acyl-l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-β-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme−product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.

Mammalian MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH2-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH2-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP+-dependent CH2-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH2-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH2-THF to formate in adult mammalian mitochondria.

A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-γ-lactonase from Bacillus thuringiensis.

Autoinducer inactivator A (AiiA) is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity but shows a preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and the F107W mutation results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrates N-acyl chain. Two aromatic residues, F64 and F68, form a hydrophobic clamp, centered around the seventh carbon in the product-bound structures decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox-sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics.

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues

Background: Mitochondria produce one-carbon units for cytoplasmic nucleotide and methyl group synthesis. Results: MTHFD2L uses both NAD+ and NADP+ and is expressed in embryonic tissues during neural tube closure. Conclusion: This cofactor specificity allows for rapid response to changing metabolic conditions. Significance: These findings help explain why mammals possess two distinct mitochondrial isozymes that switch expression during neural tube closure. Mammalian mitochondria are able to produce formate from one-carbon donors such as serine, glycine, and sarcosine. This pathway relies on the mitochondrial pool of tetrahydrofolate (THF) and several folate-interconverting enzymes in the mitochondrial matrix. We recently identified MTHFD2L as the enzyme that catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. We show here that the MTHFD2L enzyme is bifunctional, possessing both CH2-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities. The dehydrogenase activity can use either NAD+ or NADP+ but requires both phosphate and Mg2+ when using NAD+. The NADP+-dependent dehydrogenase activity is inhibited by inorganic phosphate. MTHFD2L uses the mono- and polyglutamylated forms of CH2-THF with similar catalytic efficiencies. Expression of the MTHFD2L transcript is low in early mouse embryos but begins to increase at embryonic day 10.5 and remains elevated through birth. In adults, MTHFD2L is expressed in all tissues examined, with the highest levels observed in brain and lung.

Enzymic disruption of N-aroyl-L-homoserine lactone-based quorum sensing.

Many Gram-negative bacteria use N-substituted-l-homoserine lactones as quorum-sensing signals. Naturally occurring Nsubstitutions of homoserine lactone (HSL) include acyl chains of varying length (4 to 18 carbons) and can include 3-oxo or 3hydroxy substitutions, methyl branches, and even unsaturated olefins. Structurally related synthetic N-phenylacetanoyl-HSLs and other compounds have also been shown to serve as quorum-sensing antagonists or agonists. Recently, a new class of signal, the N-aroyl-HSLs, were discovered in the Alphaproteobacterium Rhodopseudomonas palustris, which produces p-coumaroyl-l-homoserine lactone (pC-HSL). This unusual signal is especially notable because of its integration of input signals from two different kingdoms of life, bacterial cell density and plant lignin degradation products, and also because of its incorporation of an exogenous organic acid rather than endogenous intermediates of fatty-acid synthesis. This finding raises the possibility that a wide range of diverse Nsubstituted HSL signals might be used in nature. One powerful methodology used to investigate N-acyl-HSL (AHL)-mediated signaling has been the use of genetically encodable quorum-quenching enzymes that catabolize signaling molecules. However, no enzymes that abrogate pC-HSL signaling have been reported. In AHL-quenching applications, the cephalosporin acylase-related AHL acylases appear to be selective for N-acyl substitutions of particular lengths, and do not appear to specifically recognize the HSL ring. In contrast, the metallo-b-lactamase-related AHL lactonases appear to be specific for the HSL ring rather than the N substituent. For example, the AHL lactonase from Bacillus thuringiensis (AiiA) undergoes side-chain rearrangements that allow the enzyme to specifically clamp down over the lactone ring, but uses weak multivalent interactions distributed along a broad surface to bind the N-acyl substituent. These observations suggest that AiiA could be capable of recognizing and processing alternative HSL signals bearing diverse N substitutions. Here we investigate the use of AiiA to disrupt N-aroyl-HSL-based signaling (Scheme 1). To qualitatively investigate quorum-quenching of N-aroylHSL signaling by AiiA, a bioassay reported by Schaefer et al. was adapted to an agar-plate-based format. Briefly, Pseudomonas aeruginosa MW1 harbors a plasmid encoding both the pC-HSL-dependent RpaR transcription factor and a lacZ gene under the control of the rpaI promoter (pRpaR-PrpaI). Exposure to exogenous pC-HSL induces the expression of b-galactosidase, the activity of which can be visualized by using X-gal. The reporter strain and synthetic pC-HSL are incorporated into LB agar plates, into which sample wells are bored and subsequently filled with AiiA or control solutions. Quenching of pCHSL signaling by AiiA is expected to result in a halo that lacks the blue color indicative of b-galactosidase activity. Wells filled with only buffer show no appreciable effect, but treatment with AiiA results in an obvious halo (Figure 1 and

Mitochondrial one-carbon metabolism and neural tube defects

BACKGROUND Neural tube defects (NTDs) are one of the most common birth defects in humans. Maternal intake of folic acid was linked to prevention of NTDs in the 1970s. This realization led to the establishment of mandatory and/or voluntary food folic acid fortification programs in many countries that have reduced the incidence of NTDs by up to 70% in humans. Despite 40 years of intensive research, the biochemical mechanisms underlying the protective effects of folic acid remain unknown. RESULTS Recent research reveals a role for mitochondrial folate-dependent one-carbon metabolism in neural tube closure. CONCLUSION In this article, we review the evidence linking NTDs to aberrant mitochondrial one-carbon metabolism in humans and mouse models. The potential of formate, a product of mitochondrial one-carbon metabolism, to prevent NTDs is also discussed.

Human mitochondrial MTHFD2 is a dual redox cofactor-specific methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase

BackgroundFolate-dependent one-carbon metabolism provides one-carbon units for several biological processes. This pathway is highly compartmentalized in eukaryotes, with the mitochondrial pathway producing formate for use in cytoplasmic processes. The mitochondrial enzyme MTHFD2 has been reported to use NAD+ as a cofactor while the isozyme MTHFD2L utilizes NAD+ or NADP+ at physiologically relevant conditions. Because MTHFD2 is highly expressed in many cancer types, we sought to determine the cofactor preference of this enzyme.ResultsKinetic analysis shows that purified human MTHFD2 exhibits dual redox cofactor specificity, utilizing either NADP+ or NAD+ with the more physiologically relevant pentaglutamate folate substrate.ConclusionThese results show that the mitochondrial folate pathway isozymes MTHFD2 and MTHFD2L both exhibit dual redox cofactor specificity. Our kinetic analysis clearly supports a role for MTHFD2 in mitochondrial NADPH production, indicating that this enzyme is likely responsible for mitochondrial production of both NADH and NADPH in rapidly proliferating cells.

Deletion of the neural tube defect-associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion–induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.