Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T₁ has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(−)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(−)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.     

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.     

Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122.

Alcaligenes faecalis AE122 that used poly(3-hydroxybutyrate) (PHB) as a sole source of carbon was newly isolated from a coastal seawater sample. The strain required seawater for growth on PHB as well as in a nutrient broth, in which seawater could be replaced by an appropriate concentration of NaCl. PHB depolymerase was purified to homogeneity from the culture supernatant of A. faecalis AE122 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme consisted of a monomer subunit with a molecular mass of 95.5 kDa. The N-terminal amino acid sequence was GAWQNNLAGGFNKV. The dimeric and trimeric esters of 3-hydroxybutyrate were the main hydrolysis products of the purified enzyme. The enzyme was most active at pH 9.0 and 55 degrees C and was inhibited by phenylmethylsulfonyl fluoride. Several cations in seawater greatly enhanced the enzyme activity.     

Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1

A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734 bp and encodes 578 amino acids with a molecular mass of 61,757 Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.     

Structure and enzymatic degradation of poly(3-hydroxybutyrate) copolymer single crystals with an extracellular PHB depolymerase from Alcaligenes faecalis T1.

Lamellar single crystals of four random copolymers of (R)-3-hydroxybutyrate with different hydroxyalkanoates: poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyvalerate) (P(3HB-co-8%-3HV)), poly(3-hydroxybutyrate-co-10 mol%-4-hydroxybutyrate) (P(3HB-co-10%-4HB)), poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyhexanoate) (P(3HB-co-8%-3HH)) and poly(3-hydroxybutyrate-co-10 mol%-6-hydroxyhexanoate) (P(3HB-co-10%-6HH)), were grown from dilute solutions of chloroform and ethanol. All single crystals have lath-shaped morphology and the second monomer units seem to be excluded from the P(3HB) crystal, on the basis of the electron diffraction diagrams. The enzymatic degradation of P(3HB-co-8%-3HH) and P(3HB-co-10%-6HH) single crystals was investigated with an extracellular PHB depolymerase from Alcaligenes faecalis T1. Adsorption of an extracellular PHB depolymerase, examined using an immuno-gold labelling technique, demonstrated a homogeneous distribution of enzyme molecules with a low concentration on the crystal surfaces. Enzymatic degradation of single crystals progressed from the edges and ends of crystals to yield narrow cracks along their long axes and the small crystal fragments. Lamellar thicknesses of single crystals and molecular weights of copolymer chains remained unchanged during the enzymatic hydrolysis. The above results support the hypothesis that the hydrophobic adsorption of the enzyme contributes to increase the mobility of molecular chains of single crystals and generate the disordered chain-packing regions. The active-site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edges and ends with endo-exo enzymatic hydrolysis behaviour, termed processive degradation.     

Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B.

A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe. The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295.     

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.     

Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene.

The Escherichia coli K-12 ackA gene, which encodes an acetate kinase, was cloned. The acetate kinase activities of ackA+ plasmid-containing strains were amplified 160- to 180-fold. The complete nucleotide sequence of the ackA gene was determined. It was deduced that the ackA gene coded for a protein of 400 amino acids with an Mr of 43,297. The ackA gene was found to be located about 15 kilobases upstream of the purF-folC-hisT region of the chromosome.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis.

The phospholipase D (PLD) gene from Corynebacterium pseudotuberculosis has been cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data reveals a major open reading frame encoding a 31.4-kilodalton protein, a size consistent with that estimated for the PLD protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of these data with the amino-terminal protein sequence indicates that the mature PLD protein is preceded by a 24-residue signal sequence. Expression of the PLD gene in E. coli is initiated from the corynebacterial promoter, and the resulting protein has sphingomyelinase activity. Primer extension mapping localized the 5' end of the PLD gene mRNA to a site 5 to 7 base pairs downstream of a region similar to the consensus sequence for E. coli promoters. Northern and Southern blot analyses suggest that the gene is transcribed from mRNA approximately 1.1 kilobases in length and that it is present in a single copy within the C. pseudotuberculosis genome.     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.     

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.     

Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B.

The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.     

Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus.

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.     

Cloning and nucleotide sequence of the aspartase gene of Escherichia coli W.

The aspA gene of Escherichia coli W which encodes aspartase was cloned into the plasmid vector pBR322. The nucleotide sequences of aspA and its flanking regions were determined. The aspA gene encodes a protein with a molecular weight of 52,224 consisted of 477 amino acid residues. The amino acid sequence of the protein predicted from the nucleotide sequence was consistent with those of the NH2- and COOH-terminal regions and also with the amino acid composition of the purified aspartase determined previously. Potential promoter and terminator sequences for aspA were also found in the determined sequence.     

Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli.

Pantothenate permease, the product of the panF gene, catalyzes the sodium-dependent uptake of extracellular pantothenate. The panF gene was isolated from an Escherichia coli genomic DNA library and subcloned into multicopy plasmids. Increased copy number of the panF+ allele resulted in increased rates of pantothenate uptake and a significant increase in the steady-state intracellular pantothenate concentration. Despite the higher levels of pantothenate, the utilization of pantothenate for coenzyme A formation was not elevated, indicating that pantothenate kinase activity is the dominant regulator of coenzyme A biosynthesis. DNA sequencing of the panF gene revealed the presence of a single open reading frame that encoded a hydrophobic protein with a molecular weight of 51,992. Sequence analysis predicts that pantothenate permease is an integral membrane protein possessing 12 hydrophobic membrane-spanning domains connected by short hydrophilic sequences. The predicted topological profile of pantothenate permease is similar to that of other membrane carriers that catalyze cation-dependent symport.     

Histamine N-methyltransferase from rat kidney. Cloning, nucleotide sequence, and expression in Escherichia coli cells.

Complementary DNA clones encoding rat kidney histamine N-methyltransferase have been isolated using synthetic oligonucleotide probes based on partial amino acid sequences of tryptic peptides of the purified enzyme. The 1.3-kilobase cDNA consisted of a 5'-noncoding region of 8 nucleotides, a coding region of 885 nucleotides, and a 3'-noncoding region of 369 nucleotides. The encoded protein of 295 amino acid residues had a calculated molecular weight of 33,940.2. After introduction of a prokaryotic expression vector containing the isolated cDNA, Escherichia coli cells expressed histamine N-methyltransferase activity. The enzyme expressed in these cells was isolated and purified as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whose mobility was identical to the natural enzyme purified from rat kidney. The recombinant enzyme had Vmax and Km values for both histamine and S-adenosylmethionine identical to those of the natural enzyme. All of the inhibitors of the natural enzyme tested showed similar Ki values on both recombinant and natural enzyme.     

Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli.

The Escherichia coli gene firA, previously reported to code for a small, histonelike DNA-binding protein, has been cloned and found to reside immediately downstream from skp, a gene previously identified as the firA locus. firA encodes a 36-kDa protein. The mutant firA200(Ts) allele was also cloned and shown to contain three mutations, each mutation giving rise to a single amino acid change. Partially purified wild-type FirA (from a firA+ strain) and mutant FirA [from a firA200(Ts) strain] proteins have amino-terminal sequences predicted from their common DNA sequences. Both proteins lack an N-terminal methionine. Modest overexpression of wild-type or mutant FirA restored wild-type growth to firA200(Ts) strains at 43 degrees C, whereas high-level expression of wild-type FirA was required for more complete suppression of the rifampin sensitivity of firA200(Ts) rpoB double mutants. High-level expression of mutant FirA did not suppress this rifampin sensitivity.     

Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli

The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.