Development and application of a screening assay for glycoside phosphorylases

Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme’s substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl β-glucosides and phenyl β-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.     

Engineering of cellobiose phosphorylase for glycoside synthesis

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes.     

Engineering of cellobiose phosphorylase for glycoside synthesis

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes.     

Crystal structure of β‐glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides

The β‐glucosidase TnBgl1A catalyses hydrolysis of O‐linked terminal β‐glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3‐glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure‐function relationships, the crystal structure of the nucleophile mutant TnBgl1A‐E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin‐3‐glucoside (Q3), as well as for the model substrate para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3‐glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid‐3‐glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872–884. © 2016 Wiley Periodicals, Inc.     

Enzymatic synthesis of β‐glucosylglycerol using a continuous‐flow microreactor containing thermostable β‐glycoside hydrolase CelB immobilized on coated microchannel walls

β‐Glucosylglycerol (βGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable β‐glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of βGG under conditions of continuous flow at 70°C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 µL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2‐nitrophenyl‐β‐D ‐glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25–1.0 M), and the average residence time (0.2–90 s). Glycerol caused a concentration‐dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of βGG were ≥80% and ≈60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (≥80%), giving about 120 mM (30 g/L) of βGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1‐O‐β‐D ‐glucopyranosyl‐rac‐glycerol (79%) and 2‐O‐β‐D ‐glucopyranosyl‐sn‐glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme. Biotechnol. Bioeng. 2009;103: 865–872. © 2009 Wiley Periodicals, Inc.     

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.     

Carbohydrate synthesis by disaccharide phosphorylases: reactions, catalytic mechanisms and application in the glycosciences

Disaccharide phosphorylases are glycosyltransferases (EC 2.4.1.α) of specialized carbohydrate metabolism in microorganisms. They catalyze glycosyl transfer to phosphate using a disaccharide as donor substrate. Phosphorylases for the conversion of naturally abundant disaccharides including sucrose, maltose, α,α-trehalose, cellobiose, chitobiose, and laminaribiose have been described. Structurally, these disaccharide phosphorylases are often closely related to glycoside hydrolases and transglycosidases. Mechanistically, they are categorized according the stereochemical course of the reaction catalyzed, whereby the anomeric configuration of the disaccharide donor substrate may be retained or inverted in the sugar 1-phosphate product. Glycosyl transfer with inversion is thought to occur through a single displacement-like catalytic mechanism, exemplified by the reaction coordinate of cellobiose/chitobiose phosphorylase. Reaction via configurational retention takes place through the double displacement-like mechanism employed by sucrose phosphorylase. Retaining α,α-trehalose phosphorylase (from fungi) utilizes a different catalytic strategy, perhaps best described by a direct displacement mechanism, to achieve stereochemical control in an overall retentive transformation. Disaccharide phosphorylases have recently attracted renewed interest as catalysts for synthesis of glycosides to be applied as food additives and cosmetic ingredients. Relevant examples are lacto-N-biose and glucosylglycerol whose enzymatic production was achieved on multikilogram scale. Protein engineering of phosphorylases is currently pursued in different laboratories with the aim of broadening the donor and acceptor substrate specificities of naturally existing enzyme forms, to eventually generate a toolbox of new catalysts for glycoside synthesis.     

Identification of the catalytic residue of Thermococcus litoralis 4-alpha-glucanotransferase through mechanism-based labeling

Thermococcus litoralis 4-alpha-glucanotransferase (TLGT) belongs to family 57 of glycoside hydrolases and catalyzes the disproportionation and cycloamylose synthesis reactions. Family 57 glycoside hydrolases have not been well investigated, and even the catalytic mechanism involving the active site residues has not been studied. Using 3-ketobutylidene-beta-2-chloro-4-nitrophenyl maltopentaoside (3KBG5CNP) as a donor and glucose as an acceptor, we showed that the disproportionation reaction of TLGT involves a ping-pong bi-bi mechanism. On the basis of this reaction mechanism, the glycosyl-enzyme intermediate, in which a donor substrate was covalently bound to the catalytic nucleophile, was trapped by treating the enzyme with 3KBG5CNP in the absence of an acceptor and was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after peptic digestion. Postsource decay analysis suggested that either Glu-123 or Glu-129 was the catalytic nucleophile of TLGT. Glu-123 was completely conserved between family 57 enzymes, and the catalytic activity of the E123Q mutant enzyme was greatly decreased. On the other hand, Glu-129 was a variable residue, and the catalytic activity of the E129Q mutant enzyme was not decreased. These results indicate that Glu-123 is the catalytic nucleophile of TLGT. Sequence alignment of TLGT and family 38 enzymes (class II alpha-mannosidases) revealed that Glu-123 of TLGT corresponds to the nucleophilic aspartic acid residue of family 38 glycoside hydrolases, suggesting that family 57 and 38 glycoside hydrolases may have had a common ancestor.     

Glucosyl transferase activity of bovine galactosyl transferase

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glycosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor.Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.     

High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition

The diversity in substrate recognition spectra exhibited by various β‐lactamases can result from one or a few mutations in the active‐site area. Using Escherichia coli TEM‐1 β‐lactamase as a template that efficiently hydrolyses penicillins, we performed site‐saturation mutagenesis simultaneously on two opposite faces of the active‐site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended‐spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. In vivo survival assays and in vitro characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (K_M) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (k_cat) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM‐1 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.     

Multi-enzymatic glucosylation using Eucalyptus UDP-glucosyltransferase coupled UDPglucose-fermentation by bakers' yeast.

The enzymatic synthesis of glucoside compounds using a membrane-associated UDP-glucosyltransferase fraction from Eucalyptus perriniana cultured cells as a water-insoluble catalyst (N. Nakajima, et. al., J. Ferment. Bioeng., 84 (5), pp. 455-460, 1997) has been effectively done by coupling UDPglucose-fermentation by bakers' yeast. For example, beta-thujaplicin (hinokitiol) and p-aminobenzoic acid were converted respectively to their corresponding beta-D-monoglucosides with the conversion rate of around 24-26% by the multi-enzymatic system with UDPglucose as a glucose donor, which is produced by yeast cells from glucose and 5'-UMP. Addition of either cellobiose, a substrate of beta-glucosidase, or DL-1,2-anhydro-myo-inositol, an inhibitor for the enzyme in the reaction mixture, could increased the yield of these beta-D-monoglucosides. This new enzymatic system could also be used for the synthesis of flavonoid glucosides such as isoquercitrin (quercetin 3-O-beta-D-glucoside).     

Structural dissection of the reaction mechanism of cellobiose phosphorylase

Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 A (1 A=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.     

Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana

The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.     

Characterization of β-galactoside phosphorylases with diverging acceptor specificities

Glycoside phosphorylases are a special group of carbohydrate-active enzymes, with characteristics in between those of glycoside hydrolases and glycosyl transferases. The phosphorylases from family GH-112 are exceptional because they employ galactose-1-phosphate instead of glucose-1-phosphate as glycosyl donor. Different acceptor specificities have been observed in this family, ranging from l-rhamnose to GlcNAc, GalNAc and a combination of the latter. Three new phosphorylases from previously unexplored branches of the phylogenetic tree of family GH-112 have now been characterized to shed more light on this divergence in acceptor specificity. The enzymes from Erysipelothrix rhusiopathiae and Streptobacillus moniliformis were found to prefer GalNAc as acceptor, while that from Anaerococcus prevotii displays similar activities on GalNAc and GlcNAc. These results confirm the correlation between the amino acid residue at position 162 and the enzyme's specificity, i.e. a threonine in the former group and a valine in the latter. However, mutagenesis of residue 162 did not allow the rational transformation of the substrate preference, as the substitution of valine by threonine in the enzyme from Bifidobacterium longum did not tighten its specificity towards GalNAc. Unexpectedly, introducing an isoleucine at position 162 increased the preference for GlcNAc as acceptor, which illustrates that the structure-function relationships in β-galactoside phosphorylases are not yet completely understood. Several other positions have also been examined by mutational analysis but true determinants of the acceptor specificity in family GH-112 could not be identified.     

Acceptor substrate specificity of UDP-Gal: GlcNAc-R β1,3-galactosyltransferase (WbbD) from Escherichia coli O7:K1

Most of the glycosyltransferases involved in O antigen biosynthesis have not yet been characterized. We recently demonstrated that the wbbD gene of the O7 lipopolysaccharide biosynthesis cluster in E. coli strain VW187 (O7:K1) encodes WbbD, a UDP-Gal: GlcNAcα-pyrophosphate-lipid β1,3-Gal-transferase (EC 2.4.1., accession number AAC27537) that transfers the second sugar moiety in the assembly of the O7 repeating unit. The enzyme utilizes undecaprenol-pyrophosphate-GlcNAc as a natural acceptor substrate, but can also transfer Gal to GlcNAcα-PO₃-PO₃-(CH₂)₁₁-O-phenyl (GlcNAc-PP-PhU). A number of acceptor substrate analogs have now been tested to further characterize the acceptor specificity of WbbD and to determine the roles of the pyrophosphate bond and the lipid moiety in the acceptor substrate. The enzyme was found to have a low activity with a substrate containing only one phosphate group directly α-linked to GlcNAc, and the enzyme was inactive when the phosphate was absent or further removed from the anomeric carbon of GlcNAc. Modifications of the lipid chain yielded substrates with variable activities. GlcNAc derivatives that were inactive as substrates did not inhibit WbbD suggesting that these compounds did not bind to the active site of the enzyme. The specificity of mammalian β4-galactosyltransferase I has been compared to that of WbbD. The results indicate that the bacterial WbbD enzyme has a distinct specificity for GlcNAc-PP-lipid, and that WbbD recognition of its acceptor substrate is very different from that of the ubiquitous mammalian β4-galactosyltransferase I. These studies help to understand mechanisms of O antigen synthesis, to develop methods to synthesize defined oligosaccharide structures and to develop specific O antigen inhibitors.     

Structural and functional analysis of tomato β‐galactosidase 4: insight into the substrate specificity of the fruit softening‐related enzyme

Plant β‐galactosidases hydrolyze cell wall β‐(1,4)‐galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β‐galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β‐(1,4)‐galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1–7 that belongs to the β‐galactosidase/exo‐β‐(1,4)‐galactanase subfamily. The enzyme can hydrolyze a wide range of plant‐derived (1,4)‐ or 4‐linked polysaccharides, and shows a strong ability to attack β‐(1,4)‐galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β‐d ‐galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β‐sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β‐galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β‐(1,6)‐galactobiose, and ~0.6‐fold activity towards β‐(1,4)‐galactobiose, compared with wild‐type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β‐galactosidases towards β‐1,4 and β‐1,6 linkages.     

Pyranose dehydrogenases: biochemical features and perspectives of technological applications

Pyranose dehydrogenase is a fungal flavin-dependent sugar oxidoreductase which is structurally and catalytically related to fungal pyranose oxidase and cellobiose dehydrogenase and probably fulfills similar biological functions in lignocellulose breakdown. It is a monomeric secretory glycoprotein and is limited to a rather small group of litter-decomposing basidiomycetes. Compared with pyranose oxidase, it displays broader substrate specificity and a variable regioselectivity and is unable to utilize oxygen as electron acceptor using substituted benzoquinones and (organo) metallic ions instead. Depending on the structure of the sugar in pyranose form (mono/di/oligosaccharide or glycoside) and the enzyme source, selective monooxidations at C-1, C-2, C-3, or dioxidations at C-2,3 or C-3,4 of the molecule to the corresponding aldonolactones (C-1), or (di)dehydrosugars (aldos(di)uloses) can be performed. These features make pyranose dehydrogenase a promising and versatile biocatalyst for production of highly reactive, sometimes unique, di- and tri-carbonyl sugar derivatives that may serve as interesting chiral intermediates for the synthesis of rare sugars, novel drugs, and fine chemicals.     

Hydrolyses and transglycosylations performed by purified alpha-D-glucosidase of the marine mollusc Aplysia fasciata

The purification and characterisation of the alpha-glucosidase from the marine mollusc Aplysia fasciata are reported. Overall substrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied. Remarkable characteristics of this enzyme are indicated by the results of the interesting survey of transglycosylation reactions reported: pyridoxine glucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. For these last two acceptors both the yields of reactions and the concentrations of products are comparable to those obtained using glycosyl transferases; in addition, synthesis of pyridoxine and chromophoric glycosides were still possible using a 1:1 ratio maltose:acceptor which is a very interesting characteristic from a synthetic point of view (effortless purification, productivity of each reaction batch, etc.).     

Effects of cellobiose concentration on the anaerobic growth ofCellulomonas uda

Summary Anaerobic growth ofCellulomonas uda on cellobiose was investigated. With uncontrolled pH, a total metabolic inhibition due to the combined effect of low pH and acid accumulation occurred after 2 g/l cellobiose had been used. This inhibition was partly reduced when the culture pH was maintained at 7.0, but then the growth rate and growth yield were found to be both reduced. Endoglucanase synthesis was related to the cellobiose concentration under these batch growth conditions and markedly influenced by the dilution rate in chemostat.     

Substrate recognition by β-ketoacyl-ACP synthases

β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.