Preparative Synthesis of β-L-Malyl-coenzyme A Assisted by Malyl-coenzyme A Synthetase from Pseudomonas AM1

Willibald, Bertram; Boves, H.; Holler, Eggehard

doi:10.1006/abio.1995.1292

Cited by 4 publications

(3 citation statements)

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“…All attempts to measure malate activation with different nucleoside triphosphates (GTP, UTP) or CoA donors (succinyl-CoA) were unsuccessful (data not shown). Higher activities (50 mU per mg protein) have been reported using a coupled assay for ADP detection (with pyruvate kinase and lactate dehydrogenase as auxiliary enzymes) [36], and 40 mU per mg protein using an assay coupled to L -malyl-CoA lyase and spectrophotometric acetyl-CoA detection at 232 nm [29]. In all cases, the considerably lower activity compared to the postulated value (165 mU per mg) indicates that this energetically demanding reaction is not yet fully understood and could represent a metabolic bottleneck.…”

Section: Resultsmentioning

confidence: 99%

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

2010

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Background Methylobacterium extorquens AM1 is an aerobic facultative methylotrophic α-proteobacterium that can use reduced one-carbon compounds such as methanol, but also multi-carbon substrates like acetate (C2) or succinate (C4) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock.Methodology/Principal FindingsWe present a comprehensive study of all postulated enzymes for the assimilation of methanol and their regulation in response to the carbon source. Formaldehyde, which is derived from methanol oxidation, is assimilated via the serine cycle, which starts with glyoxylate and forms acetyl-CoA. Acetyl-CoA is assimilated via the proposed ethylmalonyl-CoA pathway, which thereby regenerates glyoxylate. To further the understanding of the central carbon metabolism we identified and quantified all enzymes of the pathways involved in methanol assimilation. We observed a strict differential regulation of their activity level depending on whether C1, C2 or C4 compounds are used. The enzymes, which are specifically required for the utilization of the individual substrates, were several-fold up-regulated and those not required were down-regulated. The enzymes of the ethylmalonyl-CoA pathway showed specific activities, which were higher than the calculated minimal values that can account for the observed growth rate. Yet, some enzymes of the serine cycle, notably its first and last enzymes serine hydroxymethyl transferase and malate thiokinase, exhibit much lower values and probably are rate limiting during methylotrophic growth. We identified the natural C1 carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.Conclusion/SignificanceThis study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps.

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Section: Resultsmentioning

confidence: 99%

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

2010

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“…β‐ l ‐[ 14 C]malyl‐CoA (1–5 Ci·mol −1 ) was sythesized enzymatically as described by Willibald et al . [14].…”

Section: Methodsmentioning

confidence: 99%

“…l ‐Malate in the lysates was assayed after the precipitation of proteins with 0.1 m H 2 SO 4 and neutralization of the clear supernatant with concentrated NaOH. After hydrolysis, the constituents of purified PMLA were also analysed by HPLC on Polypore H as described previously [14].…”

Section: Methodsmentioning

confidence: 99%

Is β‐poly(L‐malate) synthesis catalysed by a combination of β‐L‐malyl‐AMP‐ligase and β‐poly(L‐malate) polymerase?

Willibald

Bildl

Lee

et al. 1999

European Journal of Biochemistry

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b-Poly(l-malate) is supposed to function in the storage and transport of histones, DNA polymerases and other nuclear proteins in the giant syncytical cells (plasmodia) of myxomycetes. Here we report on the biosynthesis of [ 14 C]b-poly(l-malate) from injected l-[ 14 C]malate in the plasmodium of Physarum polycephalum. The effects of KCN, arsenate, adenosine 5 H -(a,b-methylene)triphosphate, adenosine 5 H -(b,g-methylene)triphosphate, guanosine 5 H -(b,g-methylene)triphosphate, desulfo coenzyme A and phenylarsinoxid on b-poly(l-malate) synthesis were studied after their coinjection with l-[ 14 C]malate. The synthesis was not affected by KCN or desulfo coenzyme A, but was blocked by arsenate and adenosine 5 H -(a,b-methylene)triphosphate. The plasmodium lysate catalysed an l-malate-dependent ATP-[ 32 P]pyrophosphate exchange, but was devoid of b-poly(l-malate) synthetic activity under all experimental conditions tested. The results suggested an extramitochondrial synthesis of b-poly(l-malate), involving the polymerization of b-l-malyl-AMP. It is assumed that the lack of synthesis in the lysate is caused by the inactivation of b-poly(l-malate) polymerase involving a cell injury kinase pathway. Because injected guanosine 5 H -(b,g-methylene)triphosphate blocks the synthesis, the injury signal is likely to be GTP dependent.Keywords: malyl-AMP-ligase; Physarum polycephalum; plasmodium; polymalate polymerase; polymalate synthetase.b-Poly(l-malate) (PMLA) is a polyanion with a molecular mass ranging from 5 kDa to several hundred kilodaltons and is produced by the syncytic cell form of myxomycetes ([1] and references therein). It consists of l-malyl units, which are linked by ester bonds between the b-carboxylates. The a-carboxylates are free and ionized at neutral pH. The polymer receives strong interest because of its chemical reactivity, biodegradability and availability from sustainable feedstocks [2]. As an unconventional biopolymer, it seems to have a unique physiological role, which is currently being uncovered for the plasmodium of Physarum polycephalum [3,4]. The plasmodium, which specifically produces PMLA, is a single multinuclear giant cell [1]. The nuclei divide synchronously by closed mitosis [5]. The biochemistry behind the plasmodial status is not understood, but PMLA appears to be involved.Evidence comes from the correlation of growth with PMLA synthesis, the integration of newly synthesized PMLA into nuclei [6], the high nuclear level of PMLA comparable with the levels of histones and DNA, the maintenance of a constant nuclear PMLA content, although production rates vary between wild-type and mutant strains and under different growth conditions [6], the higher order complexes of PMLA with DNA-polymerases, histones and other proteins in the nuclei [3,4,7]. It has been assumed that PMLA functions as a storage and carrier molecule to maintain an even distribution of histones, DNA polymerases and other nuclear proteins in the giant cell [4]. A plasmodium-specific protein, polymalatase, has been identified...

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Occurrence and biosynthesis of 3-mercaptopropionic acid inMethanocaldococcus jannaschii

Allen¹,

White²

2016

FEMS Microbiology Letters

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In a non-targeted analysis of thiol-containing compounds in the hyperthermophilic methanogen Methanocaldococcus jannaschii, we discovered three unexpected metabolites: 3-mercaptopropionic acid (MPA), 2-hydroxy-4-mercaptobutyric acid (HMBA) and 4-mercapto-2-oxobutyric acid (MOB). HMBA and MOB have never been reported as natural products, while MPA is highly prevalent in aquatic environments as a result of biotic and abiotic processing of sulfur-containing compounds. This report provides evidence that HMBA and MOB are part of a biosynthetic pathway to generate MPA in M. jannaschii We show that HMBA can be biosynthesized from malate semialdehyde and hydrogen sulfide, likely using a mechanism similar to that proposed for coenzyme M, coenzyme B and homocysteine biosynthesis in methanogens, where an aldehyde is converted to a thiol. The L-sulfolactate dehydrogenase, derived from the MJ1425 gene, is shown to catalyze the NAD-dependent oxidation of HMBA to MOB. Finally, we demonstrate that HMBA can be used as a biosynthetic precursor to MPA in M. jannaschii cell extracts. This proposed pathway may contribute to the wide occurrence of MPA in marine environments and indicates that MPA must serve some important function in M. jannaschii.

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Preparative Synthesis of β-L-Malyl-coenzyme A Assisted by Malyl-coenzyme A Synthetase from Pseudomonas AM1

Cited by 4 publications

References 0 publications

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

Is β‐poly(L‐malate) synthesis catalysed by a combination of β‐L‐malyl‐AMP‐ligase and β‐poly(L‐malate) polymerase?

Occurrence and biosynthesis of 3-mercaptopropionic acid inMethanocaldococcus jannaschii

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