Kieran Elborough

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen BIOCHEMICAL PROPERTIES, STRUCTURAL ANALYSIS, AND PHYLOGENETIC RELATIONSHIPS

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.

Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding.

Sequence analysis of several cDNAs encoding the phasin protein of Ralstonia eutropha indicated that the carboxyl terminus of the resulting derived protein sequence is different from that reported previously. This was confirmed by: (1) sequencing of the genomic DNA; (2) SDS‐PAGE and peptide analysis of wild‐type and recombinant phasin; and (3) mass spectrometry of wild‐type phasin protein. The results have implications for the model proposed for the binding of this protein to polyhydroxyalkanoic acid granules in the bacterium.

A Consensus Sequence for Long-chain Fatty-acid Alcohol Oxidases from Candida Identifies a Family of Genes Involved in Lipid ω-Oxidation in Yeast with Homologues in Plants and Bacteria

The yeast Candida cloacaeis capable of growing on alkanes and fatty acids as sole carbon sources. Transfer of cultures from a glucose medium to one containing oleic acid induced seven proteins of M r102,000, 73,000, 61,000, 54,000, and 46,000 and two in the region ofM r 45,000 and repressed a protein ofM r 64,000. The induction of theM r 73,000 protein reached a 7-fold maximum 24 h after induction. The protein was confirmed by its enzyme activity to be a long-chain fatty-acid alcohol oxidase (LC-FAO) and purified to homogeneity from microsomes by a rapid procedure involving hydrophobic chromatography. An internal peptide of 30 amino acids was sequenced. A 1100-base pair cDNA fragment containing the LC-FAO peptide coding sequence was used to isolate a single exon genomic clone containing the full-length coding sequence of an LC-FAO (fao1). The fao1 gene product was expressed inEscherichia coli and was translated as a functional long-chain alcohol oxidase, which was present in the membrane fraction. In addition, full-length coding sequences for a Candida tropicalis LC-FAO (faoT) and a second C. cloacae LC-FAO (fao2) were isolated. The DNA sequences obtained had open reading frames of 2094 (fao1), 2091 (fao2), and 2112 (faoT) base pairs. The derived amino acid sequences of fao2 and faoTshowed 89.4 and 76.2% similarities to fao1. Thefao1 gene is much more highly induced on alkane than isfao2. Although this study describes the first known DNA sequences encoding LC-FAOs from any source, there are unassignedArabidopsis sequences and an unassignedMycobacterium sequence in the GenBankTM Data Bank that show strong homology to the described LC-FAO sequences. The conservation of sequence between yeast, plants, and bacteria suggests that an as yet undescribed family of long-chain fatty-acid oxidases exists in both eukaryotes and prokaryotes.

Studies on wheat acetyl CoA carboxylase and the cloning of a partial cDNA

Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chromatography and electroelution. During purification bovine serum albumin (BSA) was used to coat Amicon membranes used to concentrate partially pure ACCase. Despite further SDS-PAGE/electroelution and microbore HPLC steps BSA remained associated. This presented serious protein sequencing artefacts which may reflect the affinity of BSA for fatty acids bound to ACCase. To avoid these artefacts the enzyme was digested in gel with Endoproteinase LysC protease without the presence of BSA, and the resulting peptides blotted and sequenced.A partial cDNA (1.85 kb) encoding ACCase from a wheat embryo library was cloned, which hybridised to a 7.5 kb RNA species on northern blot of wheat leaf poly(A)+ RNA. The partial cDNA therefore represents about 0.25 of the full-length cDNA. The clone was authenticated by ACCase peptide sequencing and immuno cross-reactivity of the overexpressed clone. The derived amino acid sequence showed homology with both rat and yeast ACCase sequences (62%).Antibodies raised against wheat acetyl CoA carboxylase were specific for a 220 kDa protein from both wheat embryo and leaf. In addition, by using a novel quick assay for ACCase that utilised 125I-streptavidin, we showed the major biotin containing protein to be 220 kDa in both leaf and germ. This is in marked contrast to the previously published molecular mass of 75 kDa allocated to wheat leaf ACCase.

Arabidopsis RecQsim, a plant-specific member of the RecQ helicase family, can suppress the MMS hypersensitivity of the yeast sgs1 mutant

The Arabidopsis genome contains seven genes that belong to the RecQ family of ATP- dependent DNA helicases. RecQ members in Saccharomyces cerevisiae (SGS1) and man (WRN, BLM and RecQL4) are involved in DNA recombination, repair and genome stability maintenance, but little is known about the function of their plant counterparts. The Arabidopsis thalianaRecQsim gene is remarkably different from the other RecQ-like genes due to an insertion in its helicase domain. We isolated the AtRecQsim orthologues from rice and rape and established the presence of a similar insertion in their helicase domain, which suggests a plant specific function for the insert. The expression pattern of the AtRecQsim gene was compared with the other ArabidopsisRecQ-like members in different tissues and in response to stress. The transcripts of the AtRecQsim gene were found in all plant organs and its accumulation was higher in roots and seedlings, as compared to the other AtRecQ-like members. In contrast to most AtRecQ-like genes, the examined environmental cues did not have a detectable effect on the accumulation of the AtRecQsim transcripts. The budding yeast sgs1 mutant, which is known to be hypersensitive to the DNA-damaging drug MMS, was transformed with the AtRecQsim cDNA. The AtRecQsim gene suppressed the MMS hypersensitivity phenotype of the sgs1 cells. We propose that the ArabidopsisRecQsim gene, despite its unusual structure, exhibits an evolutionary conserved function.

Acetyl-CoA Carboxylase from Brassica napus

Acetyl-CoA carboxylase (ACCase; E.C. 6.4.1.3) is a biotin containing enzyme found in all organisms and represents the first committed step towards fatty acid and flavanoid biosynthesis in higher plants. In the dicot Brassica napus , as in all other dicots studied to date, there exist two distinct forms of the enzyme [1, 2]. The Type I form has been well characterized in Arabidopsis thalina and B. napus and consists of a single polypeptide, some 220-250kDa in length. The Type II form has only recently been revealed in higher plants however. In contrast to the Type I form, Type II ACCase consists of four separate subunits, Biotin Carboxylase (BC), Biotin Carboxyl Carrier Protein (BCCP), and Carboxyl-Transferase α and β (CT α and β). This multi-subunit structure is consistent with the structure of acetyl-CoA carboxylase in prokaryotes and algae. The biotin containing protein of the Type II form has been shown to be located within purified chloroplasts from pea and oilseed rape [2]. The genetic organization of these subunits is also unique. The gene for CT β is encoded by a single gene located on the chloroplast genome whereas the other subunits are all nuclear encoded and are present as multiple copies in the Brassica genome. The genetic and multi-subunit structure of the Type II ACCase points to a complicated mechanism for co-regulation of expression of the three nuclear encoded genes, cross-talk with the chloroplast genome to regulate expression of the CT β subunit, and finally a molecular mechanism to arrange and organize the individual subunits into a complete and functional protein once translated into the chloroplast itself.

Biotin Carboxyl Carrier Protein and Biotin Carboxylase Subunits of the Multi Subunit form of Acetyl CoA Carboxylase from Brassica Napus

Plant acetyl CoA carboxylase [EC 6.4.1.3] is one of the pivotal enzymes of fatty acid biosynthesis in both seed and leaf tissue and is thought to be an important regulatory step of de nova fatty acid synthesis in chloroplasts (Post Beittenmiller et al., 1992). Its central role reflects its importance as a target for commercial herbicides. Two forms of ACCase are present in dicot plants. The chloroplast is thought to be the site for de novo fatty acid synthesis in mesophyll cells and BCCP has been shown to reside within the chloroplast. It is therefore reasonable to suppose that a type II Brassica napus ACCase is mainly associated with de novo lipid synthesis. Specific herbicides differentiate between the two forms of acetyl CoA carboxylase (ACCase) found in dicotyledonous plants.

Transgenic Modification of Acetyl CoA Carboxylase and ß-Keto Reductase Levels in Brassica Napus Functional and Regulatory Analysis

In plants de novo fatty acid biosynthesis occurs within the plastids. The first committed step in this process is the ATP dependant carboxylation of acetyl CoA to malonyl CoA. This is catalysed by acetyl CoA carboxylase (ACCase, EC 6.4.1.2), which is believed to be a key regulatory enzyme in this process (Post Beittenmiller et al. 1992). Two forms of ACCase have now been identified in dicots. A single large multifunctional polypeptide (ACCase I), and a multipeptide prokaryotic form (ACCase II). Subcellular localisation studies on ACCase II (Alban et al. 1995; Shorrosh et al. 1995; Elborough et al. 1996) indicate a chloroplast localisation. This is suggestive of a role in de novo fatty acid biosynthesis. The extrachloroplastic localisation (Alban et al 1994) of ACCase I suggests a different role. ACCase I is responsive to UV and fungal elicitors possibly indicating a role in cytosolic fatty acid elongation, and wax and flavanoid biosynthesis.

Towards a Structural Understanding of Enzymes of Lipid Biosynthesis

The biosynthesis of fatty acids is catalysed by two multienzyme systems (a) acetyl CoA carboxylase [ACC] and (b) fatty and synthetase [FAS]. We have purified the 220 KDa subunit form of ACC from wheat germ obtained amino and sequence and cloned a cDNA corresponding to the transcarboxylase domain of this enzyme[1]. The wheat cDNA was used to probe both an Arabidopsis genomic library and rape cDNA libraries prepared by oligo d(T) and random priming. A 19 Kb genomic clone has been isolated and the domain order ascribed following DNA sequencing[2].