Biomedical subjects

L Dijkhuizen

Publications and source records attributed to L Dijkhuizen.

At least 55 records · Page 3Linked to original sources

PubMed1997

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.

Adenosine Triphosphate↗

PubMed1996

The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251.

The E-domain of cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) from Bacillus circulans strain 251 is a putative raw starch binding domain. Analysis of the maltose-dependent CGTase crystal structure revealed that each enzyme molecule contained three maltose molecules, situated at contact points between protein molecules. Two of these maltoses were bound to specific sites in the E-domain, the third maltose was bound at the C-domain. To delineate the roles in raw starch binding and cyclization reaction kinetics of the two maltose binding sites in the E-domain, we replaced Trp-616 and Trp-662 of maltose binding site 1 and Tyr-633 of maltose binding site 2 by alanines using site-directed mutagenesis. Purified mutant CGTases were characterized with respect to raw starch binding and cyclization reaction kinetics on both soluble and raw starch. The results show that maltose binding site 1 is most important for raw starch binding, whereas maltose binding site 2 is involved in guiding linear starch chains into the active site. beta-Cyclodextrin causes product inhibition by interfering with catalysis in the active site and the function of maltose binding site 2 in the E-domain. CGTase mutants in the E-domain maltose binding site 1 could no longer be crystallized as maltose-dependent monomers. Instead, the W616A mutant CGTase protein was successfully crystallized as a carbohydrate-independent dimer; its structure has been refined to 2.2 A resolution. The three-dimensional structure shows that, within the error limits, neither the absence of carbohydrates nor the W616A mutation caused significant further conformational changes. The modified starch binding and cyclization kinetic properties observed with the mutant CGTase proteins thus can be directly related to the amino acid replacements.

Amino Acid Sequence↗

PubMed1996

Structure of cyclodextrin glycosyltransferase complexed with a maltononaose inhibitor at 2.6 angstrom resolution. Implications for product specificity.

Crystals of the Y195F mutant of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 were subjected to a double soaking procedure, in which they were first soaked in a solution containing the inhibitor acarbose and subsequently in a solution containing maltohexaose. The refined structure of the resulting protein-carbohydrate complex has final crystallographic and free R-factors for data in the 8-2.6 angstrom resolution range of 15.0% and 21.5%, respectively, and reveals that a new inhibitor, composed of nine saccharide residues, is bound in the active site. The first four residues correspond to acarbose and occupy the same subsites near the catalytic residues as observed in the previously reported acarbose-enzyme complex [Strokopytov et al. (1995) Biochemistry 34, 2234-2240]. An oliogosaccharide consisting of five glucose residues has been coupled to the nonreducing end of acarbose. At the fifth residue the polysaccharide chain makes a sharp turn, allowing it to interact with residues Tyr89, Phe195, and Asn193 and a flexible loop formed by residues 145-148. On the basis of the refined model of the complex an explanation is given for the product specificity of CGTases.

Amino Acid Sequence↗

PubMed1996

Crystal structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1.

The crystal structure of the cyclodextrin glycosyltransferase (CGTase) from the thermophilic microorganism Thermoanaerobacterium thermosulfurigenes EM1 has been elucidated at 2.3 A resolution. The final model consists of all 683 amino acid residues, two calcium ions and 343 water molecules, and has a crystallographic R-factor of 17.9% (Rfree 24.9%) with excellent stereochemistry. The overall fold of the enzyme is highly similar to that reported for mesophilic CGTases and differences are observed only at surface loop regions. Closer inspection of these loop regions and comparison with other CGTase structures reveals that especially loops 88-95, 335-339 and 534-539 possibly contribute with novel hydrogen bonds and apolar contacts to the stabilization of the enzyme. Other structural features that might confer thermostability to the T. thermosulfurigenes EM1 CGTase are the introduction of five new salt-bridges and three Gly to Ala/Pro substitutions. The abundance of Ser, Thr and Tyr residues near the active site and oligosaccharide binding sites might explain the increased thermostability of CGTase in the presence of starch, by allowing amylose chains to bind non-specifically to the protein. Additional stabilization of the A/E domain interface through apolar contacts involves residues Phe273 and Tyr187. No additional or improved calcium binding is observed in the structure, suggesting that the observed stabilization in the presence of calcium ions is caused by the reduced exchange of calcium from the protein to the solvent, rendering it less susceptible to unfolding. The 50% decrease in cyclization activity of the T. thermosulfurigenes EM1 CGTase compared with that of B. circulans strain 251 appears to be caused by the changes in the conformation and amino acid composition of the 88-95 loop. In the T. thermosulfurigenes EM1 CGTase there is no residue homologous to Tyr89, which was observed to take part in stacking interactions with bound substrate in the case of the B. circulans strain 251 CGTase. The lack of this interaction in the enzyme-substrate complex is expected to destabilize bound substrates prior to cyclization. Apparently, some catalytic functionality of CGTase has been sacrificed for the sake of structural stability by modifying loop regions near the active site.

Amino Acid Sequence↗

PubMed1996

Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase.

The actinomycete Amycolatopsis methanolica employs a PPi-dependent phosphofructokinase (PPi-PFK) (EC 2.7.1.90) with biochemical characteristics similar to those of both ATP- and PPi-dependent enzymes during growth on glucose. A 2.3-kb PvuII fragment hybridizing to two oligonucleotides based on the amino-terminal amino acid sequence of PPi-PFK was isolated from a genomic library of A. methanolica. Nucleotide sequence analysis of this fragment revealed the presence of an open reading frame encoding a protein of 340 amino acids with a high degree of similarity to PFK proteins. Heterologous expression of this open reading frame in Escherichia coli gave rise to a unique 45-kDa protein displaying a high level of PPi-PFK activity. The open reading frame was therefore designated pfp, encoding the PPi-PFK of A. methanolica. Upstream and transcribed divergently from pfp, a partial open reading frame (aroA) similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes was identified. The partial open reading frame (chiA) downstream from pfp was similar to chitinase genes from Streptomyces species. A phylogenetic analysis of the ATP- and PPi-dependent proteins showed that PPi-PFK enzymes are monophyletic, suggesting that the two types of PFK evolved from a common ancestor.

Actinobacteria↗

PubMed1996

Primary structure and phylogeny of the Calvin cycle enzymes transketolase and fructosebisphosphate aldolase of Xanthobacter flavus.

Xanthobacter flavus, a gram-negative facultatively autotrophic bacterium, employs the Calvin cycle for the fixation of carbon dioxide. Cells grown under autotrophic growth conditions possess an Fe(2+)-dependent fructosebisphosphate (FBP) aldolase (class II) in addition to a class I FBP aldolase. By nucleotide sequencing and heterologous expression in Escherichia coli, genes encoding transketolase (EC 2.2.1.1.; CbbT) and class II FBP aldolase (EC 4.1.2.13; CbbA) were identified. A partial open reading frame encoding a protein similar to pentose-5-phosphate 3-epimerase was identified downstream from cbbA. A phylogenetic tree of transketolase proteins displays a conventional branching order. However, the class II FBP aldolase protein from X. flavus is only distantly related to that of E. coli. The autotrophic FBP aldolase proteins from X. flavus, Alcaligenes eutrophus, and Rhodobacter sphaeroides form a tight cluster, with the proteins from gram-positive bacteria as the closest relatives.

Amino Acid Sequence↗

PubMed1996

Production of actinorhodin-related "blue pigments" by Streptomyces coelicolor A3(2).

The genetically well-known strain Streptomyces coelicolor A3(2) produces the pH indicator (red/blue) antibiotic actinorhodin, but not all the "blue pigment" produced by this strain is actinorhodin. When the organism was subjected to various nutrient limitations (ammonium, nitrate, phosphate, or trace elements), and also during growth cessation caused by a relatively low medium pH, blue pigment production was initiated but the pigment and its location varied. At pH 4.5 to 5.5, significant formation of actinorhodin occurred and was located exclusively intracellularly. At pH 6.0 to 7.5 a different blue pigment was produced intracellularly as well as extracellularly. It was purified and identified as gamma-actinorhodin (the lactone form of actinorhodin). Analysis of act mutants of S. coelicolor A3(2) confirmed that both pigments are derived from the act biosynthetic pathway. Mutants with lesions in actII-ORF2, actII-ORF3, or actVA-ORF1, previously implicated or suggested to be involved in actinorhodin export, were impaired in production of gamma-actinorhodin, suggesting that synthesis of gamma-actinorhodin from actinorhodin is coupled to its export from the cell. However, effects on the level of actinorhodin production were also found in some mutants.

Anthraquinones↗

PubMed1995

Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products.

Asp-229, Glu-257, and Asp-328 constitute the catalytic residues in cyclodextrin glycosyl transferase from Bacillus circulans strain 251. Via site-directed mutagenesis constructed D229N, E257Q, and D328N mutant proteins showed a 4,000-60,000-fold reduction of cyclization activity. A D229N/E257Q double mutant showed a 700,000-fold reduction and was crystallized for use in soaking experiments with alpha-cyclodextrin. Crystal structures were determined of wild type CGTase soaked at elevated pH with alpha-cyclodextrin (resolution, 2.1 A) and maltoheptaose (2.4 A). In addition, structures at cryogenic temperature were solved of the unliganded enzyme (2.2 A) and of the D229N/E257Q mutant after soaking with alpha-cyclodextrin (2.6 A). In the crystals soaked in alpha-cyclodextrin and maltoheptaose, a maltotetraose molecule is observed to bind in the active site. Residue 229 is at hydrogen bonding distance from the C-6 hydroxyl group of the sugar, which after cleavage will contain the new reducing end. In the D229N/E257Q double mutant structure, two alpha-cyclodextrins are observed to replace two maltoses at the E-domain, thus providing structural information on product inhibition via binding to the enzyme's raw starch binding domain.

Bacillus↗

PubMed1995

Prephenate dehydratase of the actinomycete Amycolatopsis methanolica: purification and characterization of wild-type and deregulated mutant proteins.

Prephenate dehydratase (PDT) is a key regulatory enzyme in L-phenylalanine biosynthesis in the Gram-positive bacterium Amycolatopsis methanolica. The PDT protein was purified to homogeneity (1957-fold) from wild-type cells with a final yield of 6.5%. It was characterized as a 150 kDa homotetrameric protein with a subunit size of 34 kDa. The first 35 N-terminal amino acids were identified, revealing highest similarity to the PDT proteins from Corynebacterium glutamicum and Bacillus subtilis. Kinetic studies showed that the A. methanolica PDT is allosterically inhibited by phenylalanine and activated by tyrosine. Phenylalanine caused an increase in the S0.5 for prephenate and a decrease in the Vmax. Tyrosine caused a decrease in the S0.5 for prephenate and an increase in the Vmax. Spontaneous o-fluoro- and p-fluoro-DL-phenylalanine-resistant mutants of A. methanolica were isolated. Kinetic studies with the partially purified PDT proteins of strains pFPhe32 and oFPhe84 showed that these mutant proteins had become (partly) insensitive to both phenylalanine inhibition and tyrosine activation.

Actinobacteria↗

PubMed1995

Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity.

Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 angstrum resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose. Implications for the catalytic mechanism of glycosidases.

Crystals of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans strain 251 were soaked in buffer solutions containing the pseudotetrasaccharide acarbose, a strong amylase- and CGTase inhibitor. The X-ray structure of the complex was elucidated at 2.5-A resolution with a final crystallographic R value of 15.8% for all data between 8.0 and 2.5 A. Acarbose is bound near the catalytic residues Asp229, Glu257, and Asp328. The carboxylic group of Glu257 is at hydrogen bonding distance from the glycosidic oxygen in the scissile bond between the B and C sugars (residue A is at the nonreducing end of the inhibitor). Asp328 makes hydrogen bonds with the 4-amino-4,6-dideoxyglucose (residue B), and Asp229 is in a close van der Waals contact with the C1 atom of this sugar. From this we conclude that in CGTase Glu257 acts as the proton donor and Asp229 serves as the general base or nucleophile, while Asp328 is involved in substrate binding and may be important for elevating the pKa of Glu257. On the basis of these results it appears that the absence of the C6-hydroxyl group in the B sugar is responsible for the inhibitory properties of acarbose on CGTase. This suggests that the C6-hydroxyl group of this sugar plays an essential role in the catalytic mechanism of CGTase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acarbose↗

PubMed1995

Transformation of the methylotrophic actinomycete Amycolatopis methanolica with plasmid DNA: stimulatory effect of a pMEA300-encoded gene.

Amycolatopsis methanolica contains a 13.29-kb plasmid (pMEA300) present both in the free state and integrated at a unique genomic location. A pMEA300-free derivative (strain WV1) was selected, allowing further analysis of pMEA300-encoded functions. Whole cells of strain WV1 could be transformed at high frequencies (approximately 10(6) transformants per microgram of plasmid DNA) with both circular and linear plasmid DNA, provided that the pMEA300-encoded stf (stimulation of transformation frequency) gene was present. stf would encode a putative protein of 373 amino acids with M(r) 40,201, resembling putative regulatory proteins involved in sporulation of Streptomyces griseus and Streptomyces coelicolor.

Actinobacteria↗

PubMed1995

Identification of the minimal replicon of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid (pMEA300), capable of enhancing the spontaneous mutation frequency of its host. Depending on the growth medium pMEA300 is not only maintained as an integrated element but can additionally be present as a multicopy, autonomously replicating plasmid. The minimal replicon of pMEA300 was identified. Two unlinked DNA fragments of 2.6 kb and 0.8 kb were required for pMEA300 maintenance. Sequence analysis of the 2.6 kb fragment revealed at least two open reading frames, orfA and orfB, encoding putative proteins of 170 amino acids (18,373 Da) and 416 amino acids (45,260 Da), respectively. No clear similarities were found between the deduced amino acid sequences of the putative orfA and orfB products of pMEA300 and replication proteins identified for various Streptomyces plasmids. The pMEA300 proteins of A. methanolica thus may represent unfamiliar types. The 0.8 kb fragment contained a single complete open reading frame (korA), encoding a protein of 118 amino acids (12,917 Da). The putative KorA protein of pMEA300 shows sequence similarity with various other Streptomyces plasmid-encoded Kor proteins which may belong to the GntR family of transcriptional repressor proteins. The data provide preliminary evidence for the possible involvement of a kilkor system in autonomous replication of pMEA300.

Actinobacteria↗

PubMed1995

Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1995

Cyclodextrin formation by the thermostable alpha-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase.

Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM1, recently reclassified as Thermoanaerobacterium thermosulfurigenes, clearly demonstrated that the enzyme is a cyclodextrin glycosyltransferase (CGTase). Product analysis after incubation of the enzyme with starch revealed formation of alpha-, beta-, and gamma-cyclodextrins, as well as linear sugars. The specific activity for cyclization of this CGTase was similar to those of other CGTases, whereas the specific activity for hydrolysis was relatively high in comparison with other CGTases. Alignment of the amino acid sequence of the T. thermosulfurigenes enzyme with sequences from known bacterial CGTases showed high homology. The four consensus regions of carbohydrate-converting enzymes, as well as a C-terminal raw-starch binding motif, could be identified in the sequence.

Amino Acid Sequence↗

PubMed1995

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.

Journal Article↗

PubMed1995

Fructosebisphosphatase isoenzymes of the chemoautotroph Xanthobacter flavus.

Xanthobacter flavus employs two fructosebisphosphatase (FBPase)-sedoheptulosebisphosphatase (SBPase) enzymes. One of these is constitutively expressed and has a high FBPase-to-SBPase ratio. The alternative enzyme, which is encoded by cbbF, is induced during autotrophic growth. The cbbF gene was expressed in Escherichia coli, and the FBPase was purified to homogeneity. The purified enzyme has a specific FBPase activity of 114 mumol/min/mg of protein, a Michaelis constant for fructosebisphosphate of 3 microM, and a low FBPase-to-SBPase ratio. CbbF was activated by ATP and inhibited by Ca2+.

Adenosine Triphosphate↗

PubMed1995

Identification and functional analysis of the transfer region of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica.

Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) that is present either as an integrated element or as an autonomously replicating plasmid. Conjugational transfer of pMEA300 results in pock formation, zones of growth inhibition that become apparent when plasmid-carrying donor cells develop in a confluent lawn of plasmid-lacking recipient cells. A 6.2-kb pMEA300 DNA region specifying the functions of conjugation and pock formation was sequenced, revealing 10 open reading frames. This is the first sequence of the transfer region of a plasmid from a nonstreptomycete actinomycete. No clear similarities were found between the deduced sequences of the 10 putative Tra proteins of pMEA300 and those of Streptomyces plasmids. All Tra proteins of pMEA300 thus may represent unfamiliar types. A detailed mutational analysis showed that at least four individual proteins, TraG (9,488 Da), TraH (12,586 Da), TraI (40,468 Da), and TraJ (81,109 Da), are required for efficient transfer of pMEA300. Their disruption resulted in a clear reduction in the conjugational transfer frequencies, ranging from (5.2 x 10(1))-fold (TraG) to (2.3 x 10(6))-fold (TraJ), and in reduced pock sizes. At least two putative proteins, TraA (10,698 Da) and TraB (31,442 Da), were shown to be responsible for pock formation specifically. Specific binding of the pMEA300-encoded KorA protein to the traA-korA intragenic region was observed.

Actinobacteria↗