The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.     

A Bacillus megaterium Plasmid System for the Production, Export, and One-Step Purification of Affinity-Tagged Heterologous Levansucrase from Growth Medium

A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His₆-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.     

Methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylotrophic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase

Summary Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C₅ sugars gave the highest triose accumulation (ca. 20mM after 45h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resulted in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.Offprint requests to: L. Dijkhuizen     

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.     

Biosynthesis of l-Phenylalanine and l-Tyrosine in the Actinomycete Amycolatopsis methanolica

Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.     

Identification of ATP-dependent phosphofructokinase as a regulatory step in the glycolytic pathway of the actinomycete Streptomyces coelicolor A3(2).

The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to homogeneity (1,600-fold) and characterized (110 kDa, with a single type of subunit of 40 kDa); it is allosterically inhibited by phosphoenolpyruvate. Cloning of the pfk gene of S. coelicolor A3(2) and analysis of the deduced amino acid sequence (343 amino acids; 36,667 Da) revealed high similarities to the PPi-PFK enzyme from Amycolatopsis methanolica (tetramer, nonallosteric; 70%) and to the allosteric ATP-PFK enzymes from other bacteria, e.g., Escherichia coli (tetramer; 37%) and Bacillus stearothermophilus (tetramer, 41%). Further structural and functional analysis of the two actinomycete PFK enzymes should elucidate the features of these proteins that determine substrate specificity (ATP versus PPi) and allosteric (in)sensitivity.     

Regulation of methanol metabolism in the facultative methylotroph Nocardia sp. 239 during growth on mixed substrates in batch- and continuous cultures

The regulation of methanol metabolism in Nocardia sp. 239 was investigated. Growth on mixtures of glucose or acetate plus methanol in batch cultures resulted in simultaneous utilization of the substrates. The presence of glucose, but not of acetate, repressed synthesis of the ribulose monophosphate (RuMP) cycle enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), and methanol was used as an energy source only. Comparable results were obtained following addition of formaldehyde (fed-batch system) to a culture growing on glucose. The synthesis of the methanol dissimilatory and assimilatory enzymes in Nocardia sp. 239 thus appears to be controlled differently. Methanol and/or formaldehyde induce the synthesis of these enzymes, but under carbon-excess conditions their inducing effect on HPS and HPI synthesis is completely overruled by glucose, or metabolites derived from it. Repression of the synthesis of these RuMP cycle enzymes was of minor importance under carbon- and energy-limiting conditions in chemostat cultures. Addition of a pulse of glucose to a formaldehyde-limited (2.5 mmol l^–1 h^–1) fed-batch culture resulted in a decrease in the levels of several enzymes of methanol metabolism (including HPI), whereas the HPS levels remained relatively constant. Increasing HPS/HPI activity ratios were also observed with increasing growth rates in formaldehyde-limited chemostat cultures. The data indicate that additional mechanisms, the identity of which remains to be elucidated, are involved in controlling the levels of these C₁-specific enzymes in Nocardia sp. 239.Abbreviations HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - RuMP ribulose monophosphate - FBP fructose-1,6-bisphosphate - PFK 6-phosphofructokinase     

Regulation and function of transaldolase isoenzymes involved in sugar and one-carbon metabolism in the ribulose monophosphate cycle methylotroph Arthrobacter P1

In the facultative methylotroph Arthrobacter P1 the enzyme transaldolase plays an important role in both the pentose phosphate pathway and in the ribulose monophosphate cycle of formaldehyde fixation.Among gluconate-negative mutants of Arthrobacter P1 strains occurred which also were unable to grow on xylose because they had lost the ability to synthesize transaldolase. Furthermore, this loss of transaldolase activity resulted in decreased growth rates on a number of other heterotrophic substrates. Contrary to expectation, these mutants still grew on methylamine and were even able to use gluconate as a carbon source at normal rates provided methylamine was supplied as a nitrogen source. Under these conditions high levels of transaldolase were observed.Partial purification of the transaldolases synthesized by gluconate-grown cells of wild type Arthrobacter P1 and methylamine-grown cells of one of these mutants, strain Art 98, revealed the presence of transaldolase isoenzymes. These enzymes displayed similar kinetics but were very different in heat sensitivity. Functionally these isoenzymes are apparently very similar but their synthesis is regulated differently. One of the enzymes is synthesized constitutively whereas the other is specifically induced during growth on C₁ compounds. Strain Art 98 has lost the ability to synthesize the constitutive transaldolase. It is postulated that the C₁-induced transaldolase serves to ensure a sufficiently high rate of regeneration of ribulose-5-phosphate during growth on C₁ compounds.Abbreviations RuMP ribulose monophosphate - DEAE diethylaminoethyl