Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the α-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 µmol min-1 mg-1); acarbose is a strong inhibitor of CGTase and of most other α-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of α-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the α-amylase family, changed ATase into an enzyme with 4-α-glucanotransferase activity (3.4 µmol min-1 mg-1). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 µmol min-1 mg-1).

The fully conserved Asp residue in conserved sequence region I of the alpha-amylase family is crucial for the catalytic site architecture and activity

AbstractThe α-amylase family is a large group of starch processing enzymes [Svensson, B. (1994) Plant Mol. Biol. 25, 141–157]. It is characterized by four short sequence motifs that contain the seven fully conserved amino acid residues in this family: two catalytic carboxylic acid residues and four substrate binding residues. The seventh conserved residue (Asp135) has no direct interactions with either substrates or products, but it is hydrogen-bonded to Arg227, which does bind the substrate in the catalytic site. Using cyclodextrin glycosyltransferase as an example, this paper provides for the first time definite biochemical and structural evidence that Asp135 is required for the proper conformation of several catalytic site residues and therefore for activity

The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity

Clifford Gates by Code Deformation

Topological subsystem color codes add to the advantages of topological codes an important feature: error tracking only involves measuring 2-local operators in a two dimensional setting. Unfortunately, known methods to compute with them were highly unpractical. We give a mechanism to implement all Clifford gates by code deformation in a planar setting. In particular, we use twist braiding and express its effects in terms of certain colored Majorana operators.Comment: Extended version with more detail

The remote substrate binding subsite-6 in cyclodextrin-glycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism

Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of alpha-, beta-, and gamma-cyclodextrins (cyclic alpha-(1,4)-linked oligosaccharides of 6, 7, or 8 glucose residues, respectively) from starch. Nine substrate binding subsites were observed in an x-ray structure of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite -6 is conserved in CGTases, suggesting its importance for the reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G, N193L, and G179L/G180L). All subsite -6 mutants had decreased k(cat) values for beta-cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis. Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions. The results demonstrate that (i) subsite -6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii) Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently change cyclization and coupling activities, and (iv) substrate interactions at subsite -6 activate the enzyme in catalysis via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit mechanism in the alpha-amylase family

Digestibility of resistant starch type 3 is affected by crystal type, molecular weight and molecular weight distribution

Resistant starch type 3 (RS-3) holds great potential as a prebiotic by supporting gut microbiota following intestinal digestion. However the factors influencing the digestibility of RS-3 are largely unknown. This research aims to reveal how crystal type and molecular weight (distribution) of RS-3 influence its resistance. Narrow and polydisperse α-glucans of degree of polymerization (DP) 14–76, either obtained by enzymatic synthesis or debranching amylopectins from different sources, were crystallized in 12 different A- or B-type crystals and in vitro digested. Crystal type had the largest influence on resistance to digestion (A >>> B), followed by molecular weight (Mw) (high DP >> low DP) and Mw distribution (narrow disperse > polydisperse). B-type crystals escaping digestion changed in Mw and Mw distribution compared to that in the original B-type crystals, whereas A-type crystals were unchanged. This indicates that pancreatic α-amylase binds and acts differently to A- or B-type RS-3 crystals.</p

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering

Tandem fluorescent protein timers for non-invasive relative protein lifetime measurement in plants

Targeted protein degradation is an important and pervasive regulatory mechanism in plants, required for perception and response to the environment as well as developmental signalling. Despite the significance of this process, relatively few studies have assessed plant protein turnover in a quantitative fashion. Tandem fluorescent protein timers (tFTs) offer a powerful approach for the assessment of in vivo protein turnover in distinct subcellular compartments of single or multiple cells. A tFT is a fusion of two different fluorescent proteins with distinct fluorophore maturation kinetics, which enable protein age to be estimated from the ratio of fluorescence intensities of the two fluorescent proteins. Here, we use short-lived auxin signalling proteins and model N-end rule pathway reporters to demonstrate the utility of tFTs for studying protein turnover in living plants. We present transient expression of tFTs as an efficient screen for relative protein lifetime, useful for testing the effects of mutations and different genetic backgrounds on protein stability, and demonstrate the potential for using stably expressed tFTs to study native protein dynamics with high temporal resolution in response to exogenous or endogenous stimuli

Child outcomes after placement of a cervical pessary in women with a multiple pregnancy : A 4-year follow-up of the ProTWIN trial

Acknowledgements We like to thank the research nurses of the Dutch Consortium for Healthcare Evaluation and Research in Obstetrics and Gynecology for their help in finding contact details of the parents.Peer reviewedPublisher PD

Progesterone for prevention of preterm birth in women with short cervical length : 2-year infant outcomes

ACKNOWLEDGMENTS The Triple P study is registered as NL1961. https://www.trialregister.nl/trial/1961 The original Triple P study was funded by ZonMW number 120620030. The follow-up study was funded by the Amsterdam UMC, Academic Medical Center. BWM is supported by a NHMRC Investigatorgrant (GNT1176437). BWM reports consultancy for ObsEva, Merck Merck KGaA, iGenomix and Guerbet.Peer reviewedPublisher PD