RESUMEN
Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.
Asunto(s)
Aminoácidos , Transaminasas , Citosol/metabolismo , Humanos , Cinética , Especificidad por Sustrato , Azúcares , Transaminasas/metabolismoRESUMEN
Phosphonates are compounds containing a direct carbon-phosphorus (C-P) bond, which is particularly resistant to chemical and enzymatic degradation. They are environmentally ubiquitous: some of them are produced by microorganisms and invertebrates, whereas others derive from anthropogenic activities. Because of their chemical stability and potential toxicity, man-made phosphonates pose pollution problems, and many studies have tried to identify biocompatible systems for their elimination. On the other hand, phosphonates are a resource for microorganisms living in environments where the availability of phosphate is limited; thus, bacteria in particular have evolved systems to uptake and catabolize phosphonates. Such systems can be either selective for a narrow subset of compounds or show a broader specificity. The role, distribution, and evolution of microbial genes and enzymes dedicated to phosphonate degradation, as well as their regulation, have been the subjects of substantial studies. At least three enzyme systems have been identified so far, schematically distinguished based on the mechanism by which the C-P bond is ultimately cleaved-i.e., through either a hydrolytic, radical, or oxidative reaction. This review summarizes our current understanding of the molecular systems and pathways that serve to catabolize phosphonates, as well as the regulatory mechanisms that govern their activity.
Asunto(s)
Liasas , Organofosfonatos , Humanos , Organofosfonatos/química , Liasas/genética , Bacterias/metabolismo , Fósforo/metabolismo , Fosfatos/químicaRESUMEN
The substrate specificity of enzymes is bound to be imperfect, because of unavoidable physicochemical limits. In extant metabolic enzymes, furthermore, such limits are seldom approached, suggesting that the degree of specificity of these enzymes, on average, is much lower than could be attained. During biological evolution, the activity of a single enzyme with available alternative substrates may be preserved to a significant or even substantial level for different reasons - for example when the alternative reaction contributes to fitness, or when its undesirable products are nevertheless dispatched by metabolite repair enzymes. In turn, the widespread occurrence of promiscuous reactions is a consistent source of metabolic 'messiness', from which both liabilities and opportunities ensue in the evolution of metabolic systems.
Asunto(s)
Evolución Molecular , Bioquímica , Catálisis , Especificidad por SustratoRESUMEN
N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.
Asunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Escherichia coli/enzimología , Fructosa-Bifosfato Aldolasa/química , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Periplasma/metabolismo , Conformación Proteica , Transporte de Proteínas , EstereoisomerismoRESUMEN
We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the kcat/KM values for two alternative substrates (the 'discrimination index'). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases' observed selectivities were remarkably high. In particular, GOT1 reacted ~106-fold less efficiently when the side-chain carboxylate of the 'physiological' substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using kcat/KM values to derive a global measure of enzyme specificity.
Asunto(s)
Transaminasas/química , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Aminoácidos/química , Animales , Sitios de Unión , Bovinos , Activación Enzimática , Humanos , Cinética , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Transaminasas/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5'-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW-phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW-PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.
Asunto(s)
Amoníaco-Liasas/metabolismo , Organofosfonatos/metabolismo , Vibrio/enzimología , Biocatálisis , Hidrólisis , Cinética , Organofosfonatos/química , Especificidad por SustratoRESUMEN
The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.
Asunto(s)
Fosfoglicerato-Deshidrogenasa/metabolismo , Ácidos Carboxílicos/metabolismo , Escherichia coli/metabolismo , Glicina/metabolismo , Humanos , Cinética , NAD/metabolismo , Fosfoglicerato-Deshidrogenasa/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismoRESUMEN
Steady-state enzyme kinetics typically relies on the measurement of 'initial rates', obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or 'lag phase'. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s-1.
Asunto(s)
Pruebas de Enzimas/métodos , Enzimas/química , Artefactos , Cinética , Ornitina-Oxo-Ácido Transaminasa/química , Especificidad por SustratoRESUMEN
DNAzymes (deoxyribozymes) are single-stranded DNA molecules endowed with catalytic activity, obtained by in vitro selection. In the past 25 years, dozens of DNAzymes have been identified and employed for applicative purposes, yet our knowledge of the structural and mechanistic basis of DNA catalysis remains very limited. The RNA-cleaving 8-17 DNAzyme, which depends on divalent metal ions for function, is possibly the most studied catalytic DNA in terms of mechanism. It is very efficient, implying that it adopts a combination of distinct catalytic strategies, but until recently it was uncertain which strategies are at play and how they are implemented. Recently, however, new functional studies and the attainment of high-resolution X-ray structures of an 8-17 construct, have offered a great opportunity for a more detailed understanding of its mechanism. This review examines the functional information gathered on 8-17, in the light of the available crystal structures, pointing out the congruences and possible inconsistencies between the functional and structural data. We will analyze separately three aspects of the DNAzyme function: the structural requirements for catalysis, the role of metal ions and the influence of pH on activity. Ultimately, we will contrast the experimental data with a model for the 8-17 mechanism proposed in the crystallographic study, whereby one specific G residue (G14) acts as a general base and a metal-coordinated water molecule acts as a general acid. Throughout this analysis we will signal the most outstanding mechanistic issues that remain to be addressed, with implications for the broader field of DNA catalysis.
Asunto(s)
ADN Catalítico/química , ADN Catalítico/fisiología , Animales , Cristalografía por Rayos X , ADN Catalítico/metabolismo , Guanina/química , Humanos , Metales/química , Agua/químicaRESUMEN
The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being â¼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.
Asunto(s)
Aminohidrolasas/metabolismo , Glutatión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminasas/metabolismo , Aminohidrolasas/fisiología , Animales , Desaminación , Humanos , Hidrólisis , Ratones , Ratones Noqueados , Especificidad por SustratoRESUMEN
BACKGROUND: When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. METHODS: I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). RESULTS: Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. CONCLUSIONS: The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. GENERAL SIGNIFICANCE: While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process.
Asunto(s)
Hibridación de Ácido Nucleico , Oligonucleótidos Antisentido/química , Oligonucleótidos/químicaRESUMEN
The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA substrates. The deoxyribozyme is activated by a wide variety of divalent metal ions, from Mg2+ to Pb2+, with just a few exceptions. It is not clear if metal ions are directly involved in catalysis, or are required to attain an active conformation, or both. In particular, the connection between metal-induced global structural rearrangements and catalysis is not straightforward. To gain more information on the local structural changes induced by metal ions, we introduced fluorescent 2-aminopurine (2-Ap) residues at different positions of the 8-17 'core'. We found that a construct containing 2-Ap at position 15 was best suited to monitor conformational changes in the presence of Mg2+, Ca2+ or Mn2+. Binding of these activating metal ions caused a local rearrangement at position 15, apparently entailing decreased stacking of the 2-Ap base. The metal dependence for such conformational change was generally hyperbolic (suggesting it mirrored the binding by a single metal ion) and yielded apparent dissociation constants close to those required for activation. In contrast, Cu2+, a divalent metal ion which does not support catalysis, caused in the deoxyribozyme a slow, reversible inactivation, which correlated with a very distinct conformational change at position 15.
Asunto(s)
Cobre/química , ADN Catalítico/química , Plomo/química , Magnesio/química , Manganeso/química , Cobre/metabolismo , ADN Catalítico/metabolismo , Plomo/metabolismo , Magnesio/metabolismo , Manganeso/metabolismo , Conformación ProteicaRESUMEN
The present review focuses on a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, belonging to the broader fold-type I structural group and whose archetypes can be considered ornithine δ-transaminase and γ-aminobutyrate transaminase. These proteins were originally christened "subgroup-II aminotransferases" (AT-II) but are very often referred to as "class-III aminotransferases". As names suggest, the subgroup includes mainly transaminases, with just a few interesting exceptions. However, at variance with most other PLP-dependent enzymes, catalysts in this subfamily seem specialized at utilizing substrates whose amino function is not adjacent to a carboxylate group. AT-II enzymes are widespread in biology and play mostly catabolic roles. Furthermore, today several transaminases in this group are being used as bioorganic tools for the asymmetric synthesis of chiral amines. We present an overview of the biochemical and structural features of these enzymes, illustrating how they are distinctive and how they compare with those of the other fold-type I enzymes. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
Asunto(s)
Aminas/metabolismo , Fosfato de Piridoxal/fisiología , Transaminasas/metabolismo , Sitios de Unión , Catálisis , Especificidad por Sustrato , Transaminasas/químicaRESUMEN
D-Serine is a non-proteinogenic amino acid that acts as a co-agonist of the NMDA receptors in the central nervous system. D-Serine is produced by human serine racemase (hSR), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme that also catalyzes the physiologically relevant ß-elimination of both L- and D-serine to pyruvate and ammonia. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, we found that the catalytic activity is affected by halides, in the order fluoride > chloride > bromide. On the contrary, iodide elicited a complete inhibition, accompanied by a modulation of the tautomeric equilibrium of the internal aldimine. We also investigated the reciprocal effects of ATP and malonate, an inhibitor that reversibly binds at the active site, 20 Å away from the ATP-binding site. ATP increased ninefold the affinity of hSR for malonate and malonate increased 100-fold that of ATP, confirming an allosteric interaction between the two binding sites. To further investigate this allosteric communication, we probed the active site accessibility by quenching of the coenzyme fluorescence in the absence and presence of ATP. We found that ATP stabilizes a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation.
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Adenosina Trifosfato/metabolismo , Bromuros/metabolismo , Cloruros/metabolismo , Fluoruros/metabolismo , Malonatos/metabolismo , Racemasas y Epimerasas/metabolismo , Adenosina Trifosfato/química , Sitios de Unión , Bromuros/química , Dominio Catalítico , Cloruros/química , Fluoruros/química , Humanos , Cinética , Malonatos/química , Racemasas y Epimerasas/químicaRESUMEN
Enzymes that produce or recycle folates are the targets of widely used antimalarial drugs. Despite the interest in the folate metabolism of Plasmodium falciparum, the molecular identification of ADCL (aminodeoxychorismate lyase), which synthesizes the p-aminobenzoate moiety of folate, remained unresolved. In the present study, we demonstrate that the plasmodial gene PF14_0557 encodes a functional ADCL and report a characterization of the recombinant enzyme.
Asunto(s)
Ácido Fólico/biosíntesis , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , para-Aminobenzoatos/metabolismo , Animales , Biología Computacional , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
L-Ser supply in the central nervous system of mammals mostly relies on its endogenous biosynthesis by the phosphorylated pathway (PP). Defects in any of the three enzymes operating in the pathway result in a group of neurometabolic diseases collectively known as serine deficiency disorders (SDDs). Phosphoserine phosphatase (PSP) catalyzes the last, irreversible step of the PP. Here we investigated in detail the role of physiological modulators of human PSP activity and the properties of three natural PSP variants (A35T, D32N and M52T) associated with SDDs. Our results, partially contradicting previous reports, indicate that: i. PSP is almost fully saturated with Mg2+ under physiological conditions and fluctuations in Mg2+ and Ca2+ concentrations are unlikely to play a modulatory role on PSP activity; ii. Inhibition by L-Ser, albeit at play on the isolated PSP, does not exert any effect on the flux through the PP unless the enzyme activity is severely impaired by inactivating substitutions; iii. The so-far poorly investigated A35T substitution was the most detrimental, with a 50-fold reduction in catalytic efficiency, and a reduction in thermal stability (as well as an increase in the IC50 for L-Ser). The M52T substitution had similar, but milder effects, while the D32N variant behaved like the wild-type enzyme. iv. Predictions of the structural effects of the A35T and M52T substitutions with ColabFold suggest that they might affect the structure of the flexible helix-loop region.
Asunto(s)
Dapsona/análogos & derivados , Magnesio , Monoéster Fosfórico Hidrolasas , Serina , Animales , Humanos , Serina/metabolismo , Magnesio/farmacología , Iones , Mamíferos/metabolismoRESUMEN
The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.
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Aldehído-Liasas , Fructosa-Bifosfato Aldolasa , Humanos , Animales , Ratones , Fructosa-Bifosfato Aldolasa/genética , Catálisis , Biblioteca de Genes , Glicina Hidroximetiltransferasa/genética , Carnitina , MamíferosRESUMEN
Dysregulated expression of the AGXT2L1 gene has been associated to neuropsychiatric disorders. Recently the gene product was shown to possess O-phosphoethanolamine phospho-lyase activity. We here analyze the specificity of AGXT2L1 in terms of both reaction and substrate. We show that the enzyme, despite having evolved from a transaminase ancestor, is at least 500-fold more active as a lyase than as an aminotransferase. Furthermore, the lyase reaction is very selective for O-phosphoethanolamine, strongly discriminating against closely related compounds, and we dissect the factors that contribute to such narrow substrate specificity. Overall, AGXT2L1 function appears to be rigidly confined to phospholipid metabolism, which is altered in neuropsychiatric disturbances.
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Trastorno Bipolar/enzimología , Liasas/genética , Esquizofrenia/enzimología , Transaminasas/genética , Trastorno Bipolar/genética , Trastorno Bipolar/patología , Etanolaminas/química , Etanolaminas/metabolismo , Regulación de la Expresión Génica , Humanos , Cinética , Liasas/química , Liasas/metabolismo , Esquizofrenia/genética , Esquizofrenia/patología , Especificidad por Sustrato , Transaminasas/química , Transaminasas/metabolismoRESUMEN
Organisms from all kingdoms of life synthesize L-serine (L-Ser) from 3-phosphoglycerate through the phosphorylated pathway, a three-step diversion of glycolysis. Phosphoserine aminotransferase (PSAT) catalyzes the intermediate step, the pyridoxal 5'-phosphate-dependent transamination of 3-phosphohydroxypyruvate and L-glutamate to O-phosphoserine (OPS) and α-ketoglutarate. PSAT is particularly relevant in the central nervous system of mammals because L-Ser is the metabolic precursor of D-serine, cysteine, phospholipids, and nucleotides. Several mutations in the human psat gene have been linked to serine deficiency disorders, characterized by severe neurological symptoms. Furthermore, PSAT is overexpressed in many tumors and this overexpression has been associated with poor clinical outcomes. Here, we report the detailed functional and structural characterization of the recombinant human PSAT. The reaction catalyzed by PSAT is reversible, with an equilibrium constant of about 10, and the enzyme is very efficient, with a kcat /Km of 5.9 × 106 M-1 s-1 , thus contributing in driving the pathway towards the products despite the extremely unfavorable first step catalyzed by 3-phosphoglycerate dehydrogenase. The 3D X-ray crystal structure of PSAT was solved in the substrate-free as well as in the OPS-bound forms. Both structures contain eight protein molecules in the asymmetric unit, arranged in four dimers, with a bound cofactor in each subunit. In the substrate-free form, the active site of PSAT contains a sulfate ion that, in the substrate-bound form, is replaced by the phosphate group of OPS. Interestingly, fast crystal soaking used to produce the substrate-bound form allowed the trapping of different intermediates along the catalytic cycle.
Asunto(s)
Serina , Transaminasas , Animales , Humanos , Sistema Nervioso Central/metabolismo , Mamíferos , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Serina/metabolismo , Transaminasas/químicaRESUMEN
Phosphonates-compounds containing a direct C-P bond-represent an important source of phosphorus in some environments. The most common natural phosphonate is 2-aminoethylphosphonate (AEP). Many bacteria can break AEP down through specialized "hydrolytic" pathways, which start with the conversion of AEP into phosphonoacetaldehyde (PAA), catalyzed by the transaminase PhnW. However, the substrate scope of these pathways is very narrow, as PhnW cannot process other common AEP-related phosphonates, notably N-methyl AEP (M1AEP). Here, we describe a heterogeneous group of FAD-dependent oxidoreductases that efficiently oxidize M1AEP to directly generate PAA, thus expanding the versatility and usefulness of the hydrolytic AEP degradation pathways. Furthermore, some of these enzymes can also efficiently oxidize plain AEP. By doing so, they surrogate the role of PhnW in organisms that do not possess the transaminase and create novel versions of the AEP degradation pathways in which PAA is generated solely by oxidative deamination.