- Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase
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N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N-bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains—including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site—was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.
- Gu, Zhenghua,Guo, Zitao,Shao, Jun,Shen, Chen,Shi, Yi,Tang, Mengwei,Xin, Yu,Zhang, Liang
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- Cell-free biocatalytic syntheses of l-pipecolic acid: A dual strategy approach and process intensification in flow
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As an alternative to the traditional chemical synthesis or in vivo production of l-pipecolic acid, we have developed two ex vivo strategies using purified and immobilised enzymes for the production of this key building block. Firstly, a transaminase capable of lysine ?-deamination was coupled with a novel pyrroline-5-carboxylate reductase, yielding 60% conversion at the 50 mM scale with free enzymes and in situ recycling of the cofactor. A second, simpler, redox neutral system was then constructed by combining the pyrroline-5-carboxylate reductase with a lysine-6-dehydrogenase. This bienzymatic system, with catalytic amount of free cofactor yielded >99% of pipecolic acid in batch and, following co-immobilisation of both enzymes, it was applied as a packed-bed reactor in continuous flow achieving again a molar conversion of >99% with 30 min residence time, and a space-time yield up to 2.5 g L-1 h-1. The sustainability of the system was further improved by a catch-and-release strategy to purify the product, and recovery and recycling of the cofactor.
- Benítez-Mateos, Ana I.,Calvey, Liam,Paradisi, Francesca,Roura Padrosa, David
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supporting information
p. 5310 - 5316
(2020/09/17)
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- Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from a hyperthermophilic archaeon
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The hypothetical OCC_00372 protein from Thermococcus litoralis is a member of the ProR superfamily from hyperthermophilic archaea and exhibits unique bifunctional proline racemase/hydroxyproline 2-epimerase activity. However, the molecular mechanism of th
- Watanabe, Yasunori,Watanabe, Seiya,Itoh, Yoshika,Watanabe, Yasuo
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p. 135 - 140
(2019/02/19)
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- Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes
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Cysteine S-nitrosation is a reversible post-translational modification mediated by nitric oxide (NO)-derived agents. S-Ni-trosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical NO-signaling pathway, little is understood about how much S-nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S-nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein S-nitrosation. To identify proteins whose activities are modulated by S-nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S-nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S-nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, 1-pyrroline-5-carboxylate dehydrogenase, catechol-O-methyltransferase, and D-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S-nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S-nitrosation in each enzyme.
- Bruegger, Joel J.,Smith, Brian C.,Wynia-Smith, Sarah L.,Marletta, Michael A.
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p. 6282 - 6296
(2018/05/03)
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- A unique cis -3-hydroxy- l -proline dehydratase in the enolase superfamily
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The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily (UniProt ID A0NXQ8). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to δ1-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.
- Zhang, Xinshuai,Kumar, Ritesh,Vetting, Matthew W.,Zhao, Suwen,Jacobson, Matthew P.,Almo, Steven C.,Gerlt, John A.
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supporting information
p. 1388 - 1391
(2015/03/05)
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- Kinetic and isotopic characterization of l -proline dehydrogenase from mycobacterium tuberculosis
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The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate in the proline catabolic pathway. The ProDH gene, prub, was cloned into the pYUB1062 vector, and the C-terminal His-tagged 37 kDa protein was expressed and purified by nickel affinity chromatography. A steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 s-1 and K m values of 5.7 ± 0.8 mM and 3.4 ± 0.3 μM for l-proline and 2,6-dichlorophenolindophenol (DCPIP), respectively. The pH dependence of kcat revealed that one enzyme group exhibiting a pK value of 6.8 must be deprotonated for optimal catalytic activity. Site-directed mutagenesis suggests that this group is Lys110. The primary kinetic isotope effects on V/KPro and V of 5.5 and 1.1, respectively, suggest that the transfer of hydride from l-proline to FAD is rate-limiting for the reductive half-reaction, but that FAD reoxidation is the rate-limiting step in the overall reaction. Solvent and multiple kinetic isotope effects suggest that l-proline oxidation occurs in a stepwise rather than concerted mechanism. Pre-steady-state kinetics reveal an overall kred of 88.5 ± 0.7 s-1, and this rate is subject to a primary kinetic isotope effect of 5.2. These data confirm that the overall reaction is limited by reduced flavin reoxidation in the second half-reaction.
- Serrano, Hector,Blanchard, John S.
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p. 5009 - 5015
(2013/08/23)
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- In vitro reconstitution of the first steps of anatoxin-a biosynthesis in Oscillatoria PCC 6506: From free L-proline to acyl carrier protein bound dehydroproline
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Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases for which the starter would be (S)-1-pyrroline-5-carboxylate bound to an acyl carrier protein, AnaD. We now report the in vitro reconstitution of the first steps of this biosynthesis in Oscillatoria PCC 6506. We identified in PCC 6506 the gene coding for an Sfp-like phosphopantetheinyl transferase and purified the gene product, OsPPT, that catalyzed the transfer of the phosphopantetheinyl arm to the serine 41 of AnaD. The pure adenylation protein AnaC loaded L-proline on holo-AnaD and was specific for L-proline (Km = 0.97 mM, kcat =68 min-1) among the 20 natural amino acids. Among six close structural analogues of L-proline, including (S)-1-pyrroline-5-carboxylate, we only found 3,4-dehydro-L-proline to be an alternate substrate for AnaC (Km=1.5 mM, kcat=29min-1). The putative prolyl-AnaD dehydrogenase, AnaB, purified to homogeneity as a histidine-tagged protein, showed an absorption spectrum characteristic of FAD-containing proteins. It oxidized prolyl-AnaD to dehydroprolyl-AnaD as shown by tryptic digestion of the protein followed by liquid chromatography coupled to tandem mass spectrometry. Alignment of the amino acid sequence of this dehydrogenase with related enzymes showed that AnaB belongs to the acyl-CoA dehydrogenase superfamily and thus probably catalyzes an α-β-dehydrogenation of the thioester-bound proline followed by an aza-allylic isomerization to yield (S)-pyrroline-5-carboxyl-AnaD, the proposed starter for the subsequent polyketide synthase, AnaE.
- Mejean, Annick,Mann, Stephane,Vassiliadis, Gaelle,Lombard, Berangere,Loew, Damarys,Ploux, Olivier
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experimental part
p. 103 - 113
(2010/10/21)
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- δ1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline
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Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Δ1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21-(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of L-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.
- Huang, Tzou-Chi,Huang, Yi-Wen,Hung, Hui-Ju,Ho, Chi-Tang,Wu, Mei-Li
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p. 5097 - 5102
(2008/02/12)
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- C-prolinylquercetins from the yellow cocoon shell of the silkworm, Bombyx mori
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Two flavonoids containing the L-proline moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A) and 6-C-[(2S,5R)-prolin-5-yl] quercetin (prolinalin B), were isolated from the cocoon shell of the silkworm, Bombyx mori. Their structural elucidation was achieved by application of acid hydrolysis and spectroscopic methods. These compounds were not found in the leaves of mulberry (Morus alba L.), the host plant of the silkworm, suggesting that the flavonoids are metabolites of the insect. This is the first time that flavonoids with an amino acid moiety have been found as naturally occurring compounds.
- Hirayama, Chikara,Ono, Hiroshi,Tamura, Yasumori,Nakamura, Masatoshi
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p. 579 - 583
(2008/02/05)
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- Biosynthesis of 4-methylproline in cyanobacteria: Cloning of nosE and nosF genes and biochemical characterization of the encoded dehydrogenase and reductase activities
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The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria was investigated on the genetic and enzymatic level. Two genes involved in the biosynthesis were cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a Δ1-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli and biochemically characterized. Putative substrates were synthesized to test enzyme substrate specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of the enzymatic reactions with respect to the substrates as well as to the coenzymes.
- Luesch, Hendrik,Hoffmann, Dietmar,Hevel, Joan M.,Becker, Julia E.,Golakoti, Trimurtulu,Moore, Richard E.
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