Broadening the biocatalytic properties of recombinant sucrose synthase 1 from potato (Solanum tuberosum L.) by expression in Escherichia coli and Saccharomyces cerevisiae

Sauerzapfe, Birgit; Engels, Leonie; Elling, Lothar

doi:10.1016/j.enzmictec.2008.04.001

Cited by 25 publications

(23 citation statements)

References 40 publications

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“…To ensurea ne qual distribution of UDP between the regeneration systems, PK was applied in excess over SuSy,b ecause its K m for UDP (2.6 mm) [25] is an order of magnitude lower than that of SuSy (0.2 mm). [26] The agarose gel electrophoresisa nalysis ( Figure 8C)c onfirms the formation of HA with a M w of 1.22 MDaa fter 24 hand complete UDP-sugar conversion. However,c ompared with the corresponding reaction with UDP-GlcA regenerationa lone (2.30 MDa, see Figure 7B,r eaction f), the M w of the HA polymer is reduced by 50 %.…”

Section: Evaluation Of Ems Including Udp-glca and Udp-glcnac Regeneramentioning

confidence: 71%

In Vitro One‐Pot Enzymatic Synthesis of Hyaluronic Acid from Sucrose and N‐Acetylglucosamine: Optimization of the Enzyme Module System and Nucleotide Sugar Regeneration

et al. 2018

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Enzymatic in vitro synthesis of hyaluronic acid (HA) is the technology of choice for the production of defined HA preparations under controlled conditions. One drawback is the high costs for the substrates uridine diphosphate (UDP)‐GlcA and UDP‐GlcNAc. We developed an economic multi‐enzyme strategy for the in vitro one‐pot synthesis of high‐molecular‐weight (Mw)‐HA from cheap and sustainable substrates sucrose and N‐acetylglucosamine (GlcNAc) with in situ regeneration of nucleotide sugars. Multiplexed capillary electrophoresis was used for analysis and optimization of the reaction cascades. We here present novel parameters influencing the polymerization rate and Mw of HA synthesized by Pasteurella multocida hyaluronan synthase (pmHAS1‐703‐His6), including cofactors, kinetics, and substrate ratio. UDP‐GlcA regeneration by sucrose synthase proved to be favorable for kinetic and economic reasons. With optimized reaction conditions, HA with a Mw>2 MDa is synthesized from catalytic UDP concentrations and 10 mm GlcNAc in less than 10 h.

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Section: Evaluation Of Ems Including Udp-glca and Udp-glcnac Regeneramentioning

confidence: 71%

In Vitro One‐Pot Enzymatic Synthesis of Hyaluronic Acid from Sucrose and N‐Acetylglucosamine: Optimization of the Enzyme Module System and Nucleotide Sugar Regeneration

et al. 2018

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“…Enzyme activity was measured using a continuous photometric assay in a microtiter plate as described previously 16. Furthermore, enzyme activity was determined at linear conversion rate with CE‐UV analysis of UDP‐Glc at 254 nm as described above.…”

Section: Methodsmentioning

confidence: 99%

“…The sucrose synthase (SuSy) module generates UDP‐α‐ D ‐glucose (UDP‐Glc, 1 ) from sucrose and UDP with recombinant SuSy1 (EC 2.4.1.13). from potato 16. UDP‐Glc is further converted in the second UDP‐Glc‐dehydrogenase (UGDH) module to UDP‐α‐ D ‐glucuronic acid (UDP‐GlcA, 2 ) by recombinant human UDP‐Glc‐dehydrogenase (EC 1.1.1.22).…”

Section: Introductionmentioning

confidence: 99%

Enzyme Module Systems for the Synthesis of Uridine 5′‐Diphospho‐α‐D‐glucuronic Acid and Non‐Sulfated Human Natural Killer Cell‐1 (HNK‐1) Epitope

et al. 2015

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Tailor-made strategies for the stereo-and regioselective multi-step enzymatic synthesis of glycoconjugates require well characterized glycosyltransferases andc arbohydrate modifying enzymes. We here report on an ovel enzyme cascade for the synthesiso fu ridine 5'-diphospho-a-d-glucuronic acid (UDP-GlcA) andt he non-sulfated human natural killer cell-1 (HNK-1) epitope including in situ regenerationo fU DP-GlcA andt he cofactor nicotinamide adenine dinucleotide NAD + + by the combination of four enzymes in one-pot.I nt he first enzyme module sucroses ynthase 1( SuSy1) is usedt op roduce uridine 5'-diphospho-a-d-glucose (UDP-Glc) from sucrose and uridine 5'-diphosphate (UDP). Thec ombination with UDP-Glc dehydrogenase in the second enzyme module leads to the synthesiso fU DP-GlcA with concomitant in situ regeneration of the cofactor NAD + + by nicotinamide adenine dinucleotide hydride (NADH)-oxidase.I nt he third enzyme module the mammalian glucuronyltransferase GlcAT-Pc atalyzes the synthesiso ft he non-sulfated HNK-1e pitope by regioselective transfer of GlcA onto N-acetyllactosamine type 2( LacNAc type 2). We present ac omprehensive studyo ns ubstrate kinetics,s ubstrate specificities,v ariation and relation of enzyme activitiesa s well as cross inhibition of intermediate products.With optimized reactionc onditions we obtain superior product yieldsw ith streamlined synthesisc osts for the expensiven ucleotide sugar UDP-GlcA and cofactor NAD + + .

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“…Previous studies of non-recombinant barley UGPase (purified from barley malt) ( Ritter et al, 1996 ) demonstrated that the enzyme had a somewhat less strict substrate specificity (i.e., low activity with GlcA-1-P and GalA-1-P, in addition to that with Glc-1-P), when compared to the recombinant (expressed from procaryote hosts) enzymes used in the present study. These differences in substrate specificity could perhaps be explained by post-translational modifications which are specific for eucaryotes/plants ( Sauerzapfe et al, 2008 ) and/or simply by differences in assay conditions, e.g., 1 vs. 2 mM NTP and 5 vs. 10 mM MgCl 2 for this study and Ritter’s et al, (1996 ), respectively. However, it should be emphasized that, in terms of substrate specificity, plant UGPases are very different from mammalian UGPases which, in addition to Glc-1-P, have relatively high activities with several other sugar-1-phosphates, producing corresponding UDP-sugars ( Knop and Hansen, 1970 ; Ritter et al, 1996 ).…”

Section: Discussionmentioning

confidence: 96%

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

Decker

Kleczkowski

2017

Front. Plant Sci.

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UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

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Broadening the biocatalytic properties of recombinant sucrose synthase 1 from potato (Solanum tuberosum L.) by expression in Escherichia coli and Saccharomyces cerevisiae

Cited by 25 publications

References 40 publications

In Vitro One‐Pot Enzymatic Synthesis of Hyaluronic Acid from Sucrose and N‐Acetylglucosamine: Optimization of the Enzyme Module System and Nucleotide Sugar Regeneration

In Vitro One‐Pot Enzymatic Synthesis of Hyaluronic Acid from Sucrose and N‐Acetylglucosamine: Optimization of the Enzyme Module System and Nucleotide Sugar Regeneration

Enzyme Module Systems for the Synthesis of Uridine 5′‐Diphospho‐α‐D‐glucuronic Acid and Non‐Sulfated Human Natural Killer Cell‐1 (HNK‐1) Epitope

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

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