Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate.

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5'-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2',3'-trihydroxy-3-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.

Cloning and sequencing of a second laccase gene from the white-rot fungus Coriolus versicolor.

A second laccase gene, CVLG1, was isolated from Coriolus versicolor. CVLGI encodes a precursor protein of 526 amino acids which contains a 23-amino acid signal sequence, and the coding region is interrupted by 11 introns. The number of potential N-glycosylation sites in this product is 12 and the greatest among that of polyporales laccases. Moreover, this protein shares about 70% homology with other polyporales laccases. Genomic Southern analysis showed that C. versicolor laccases are encoded by more than four genes including CVLG1 and a transposed allele of this gene.

Isolation and analysis of cinnamic acid 4-hydroxylase homologous genes from a hybrid aspen, Populus kitakamiensis.

Cinnamic acid 4-hydroxylase (CA4H) is the second enzyme involved in phenylpropanoid biosynthesis and is a member of the cytochrome P-450 superfamily. Three CA4H homologous genes, cyp73A, cyp73b, and cyp73c, and a cDNA clone of cyp73a were isolated from a genomic library and a cDNA library of a hybrid aspen; Populus kitakamiensis, and were characterized. They might be interrupted by two introns each. cyp73a and cyp73b were very similar to each other not only in coding regions but also in non-coding regions. Southern blot analysis showed that four homologous genes for CA4H constructed a small gene family in the diploid genome of P. kitakamiensis. In the promoter regions, there were many common cis-element-like sequences in phenylpropanoid biosynthesis genes.

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis.

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNA1 (Osakabe et al., 1995, Plant Sci. 105: 217-226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover, the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.

Characterization of the structure and determination of mRNA levels of the phenylalanine ammonia-lyase gene family from Populus kitakamiensis.

We isolated two new PAL genes, palg2b and palg4, from Populus kitakamiensis, palg2a and palg2b are clustered and palg2b encodes a polypeptide of 710 amino acids. The nucleotide sequence in the coding region of palg2b was 94.6% identical to that of palg2a. The promoter regions of palg1, palg2a and palg2b have several elements conserved among many phenylpropanoid biosynthetic genes. We measured the mRNA levels of the four PAL genes by S1 mapping using total RNA from stem tissues developing secondary xylem. Results showed that the transcript level of palg2b was higher than that of the other PAL genes.

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem.

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5' and 3' ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.

Characterization of the C alpha-dehydrogenase gene involved in the cleavage of beta-aryl ether by Pseudomonas paucimobilis.

C alpha-dehydrogenase catalyzes the oxidation of arylglycerol-beta-aryl ether at the C alpha-position, and therefore this process produces the specific substrate for beta-etherase, which cleaves beta-ary ethers (C alpha carbonyl type). Here we isolated the C alpha-dehydrogenase gene (lig D) and sequenced its nucleotides. This gene contains an open reading frame of 915 bp and the deduced amino acid sequence had a homology with the ribitol dehydrogenase family. lig D is about 1 kbp upstream of the beta-etherase gene (lig E).

A bacterial enzyme degrading the model lignin compound beta-etherase is a member of the glutathione-S-transferase superfamily.

Cleavage of beta-aryl ether linkages is essential in lignin degradation. We identified another beta-etherase gene (ligF), which contains an open reading frame of 771 bp and lies between genes coding C alpha-dehydrogenase (ligD) and beta-etherase (ligE). The beta-etherase activity of LigF expressed in Escherichia coli was more than 80 times as high as that of LigE. ligF and ligE are homologous to glutathione-S-transferase, and upon addition of glutathione a remarkable acceleration of beta-etherase activity was found in E. coli carrying ligF. It is concluded that LigF plays a central role in beta-aryl ether cleavage and that glutathione is the hydrogen donor in this reaction.

Cloning and sequencing of the gene for a Pseudomonas paucimobilis enzyme that cleaves beta-aryl ether.

We isolated Pseudomonas paucimobilis SYK-6, which was able to degrade various dimeric lignin compounds (Y. Katayama, S. Nishikawa, M. Nakamura, K. Yano, M. Yamasaki, N. Morohoshi, and T. Haraguchi, Mokuzai Gakkaishi 33:77-79, 1987). This metabolic process is a distinct characteristic of this bacterium, which is equipped with an enzymatic modification system for various dimeric lignin compounds involved in the tricarboxylic acid cycle. Cleavage of the beta-aryl ether linkage is essential in this process, because this linkage is the most abundant (approximately 50%) in lignin. Here, we report the isolation and characterization of the beta-etherase gene, which contains an open reading frame of 843 bp and which we call ligE. This gene was expressed in Escherichia coli, and the enzyme had the same kinetic properties as the P. paucimobilis SYK-6 enzyme.

Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.

Detection and localization of a new enzyme catalyzing the beta-aryl ether cleavage in the soil bacterium (Pseudomonas paucimobilis SYK-6).

Cleavage of the arylglycerol-beta-aryl ether linkage is the most important process in the biological degradation of lignin. We determined the activity of the enzyme cleaving the beta-aryl ether linkage in membranes of Pseudomonas paucimobilis SYK-6. This enzyme was tightly associated with the cellular membrane and catalyzed the unique and reductive cleavage of compound II but not cleavage of compound I. This enzymatic activity was stimulated by addition of NADH. On the basis of this evidence, we present a model of the specific cellular assimilation of beta-aryl ether by P. paucimobilis SYK-6.