Reductive, Coenzyme A-Mediated Pathway for 3-Chlorobenzoate Degradation in the Phototrophic Bacterium Rhodopseudomonas palustris

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl–CoA), (ii) reductively dehalogenating 3-chlorobenzoyl–CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.     

At4g24160, a Soluble Acyl-Coenzyme A-Dependent Lysophosphatidic Acid Acyltransferase

Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the α/β-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX₄D acyltransferase motif, and V(X)₃HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.Acylation of glycerol-3-phosphate (G3P) is the first step in the biosynthesis of glycerolipids in plants. Most of the enzymes involved in this pathway were shown to be membrane bound (Somerville and Browse, 1991). However, a soluble G3P acyltransferase has been reported in plants, which acylates G3P to lysophosphatidic acid (LPA) in an acyl-(acyl carrier protein)-dependent manner (Murata and Tasaka, 1997). The role of other soluble enzymes in the glycerolipid biosynthesis pathway is well documented. Cytosolic monoacylglycerol acyltransferase (Tumaney et al., 2001), diacylglycerol acyltransferase (Saha et al., 2006), and LPA phosphatase (Shekar et al., 2002) were shown to be present in the immature seeds of Arachis hypogaea. Recently, cytosolic LPA phosphatase (Reddy et al., 2008) and phosphatidic acid (PA) phosphatase have also been reported in Saccharomyces cerevisiae (Han et al., 2006). In addition, we demonstrated earlier the presence of a soluble LPA acyltransferase (LPAAT) as a part of the cytosolic multienzyme complex for the synthesis of triacylglycerol (TG) in Rhodotorula glutinis (Gangar et al., 2001). In S. cerevisiae, Ict1p catalyzes the acylation of LPA to PA, thereby enhancing phospholipid biosynthesis under cellular stress. A Δict1 deletion strain was shown to be calcofluor white sensitive and exhibited a defective phospholipid biosynthesis, suggesting a role of Ict1p in the maintenance of the cell membranes (Ghosh et al., 2008a).BLAST analysis of the human genome with the Ict1p sequence resulted in the identification of a gene named CGI-58. Mutations in human CGI-58 are responsible for a rare autosomal recessive genetic disorder known as Chanarin Dorfman syndrome (Zechner et al., 2009). CGI-58 is a member of the α/β-hydrolase family of proteins and has a conserved lipase motif GXNXG, where the Ser is replaced by an Asn. Biochemical characterization of human CGI-58 revealed that it acylates LPA to PA. Heterologous overexpression in yeast showed that expression of CGI-58 enhanced the biosynthesis of total phospholipids, especially PA, phosphatidylethanolamine, and phosphatidylcholine (PC). CGI-58 was found to localize to the lipid bodies isolated from the mice white adipose tissues, but the LPAAT activity in the soluble fraction from adipose tissue was also attributed to CGI-58 (Ghosh et al., 2008b).So far, a soluble LPAAT from plants has not been identified, although the importance of such enzymes in other experimental systems has been envisaged (Tumaney et al., 2001; Ghosh et al., 2008a). Being aware of the important role of Ict1p and CGI-58 in phospholipid metabolism and in combating stress, we started a systematic search for CGI-58-like proteins in plants. The availability of the complete genome sequence of Arabidopsis (Arabidopsis thaliana) allowed us to perform a comprehensive genome-wide survey of CGI-58 like proteins in Arabidopsis. As will be described in this study, a BLAST analysis of CGI-58 in Arabidopsis revealed At4g24160 as its closest homolog. Biochemical characterization of At4g24160 showed its ability to acylate LPA to PA in an acyl-CoA-dependent manner. The recombinant protein has the capability to hydrolyze TG and PC to a lesser extent. Expression analysis of At4g24160 and its homologs suggests the significance of these genes under various stress conditions. In summary, At4g24160 is a soluble acyltransferase with lipase and phospholipase functions from Arabidopsis belonging to the α/β-hydrolase superfamily of proteins.     

Coupled Metabolic and Photolytic Pathway for Degradation of Pyridinedicarboxylic Acids, Especially Dipicolinic Acid

Three isomers of pyridinedicarboxylic acid (PDCA) (2,3-, 2,5-, and 2,6-PDCA) were partially oxidized by marine bacteria when grown aerobically on the corresponding phthalate analogs. The metabolites, unlike the parent PDCAs, absorbed light in the solar actinic range (wavelengths greater than 300 nm) and were readily degraded in sunlight. The principal product from 2,6-PDCA (dipicolinic acid) metabolism was extracted from a culture fluid, purified by column chromatography, and analyzed by UV-visible, infrared, and ¹³C nuclear magnetic resonance spectroscopy. The compound was identified as 2,3-dihydroxypicolinic acid (2,3-DHPA). 2,3-DHPA was photolyzed in aqueous solution (pH 8.0) with a half-life of 100 min. Eight photoproducts, three of which were photolabile, were detected by high-performance liquid chromatography. Ammonia was also photoproduced from 2,3-DHPA. Analysis of the photoproducts by UV-visible spectroscopy and by high-performance liquid chromatography of 2,4-dinitrophenylhydrazones indicated that the products were conjugated carbonyls and carboxylic acids. Six of the photoproducts were readily consumed by bacterial strain CC9M. In illuminated aquatic environments, coupled bio- and photodegradative mechanisms probably contribute to the degradation of PDCAs.     

Sequence Analysis of the GntII (Subsidiary) System for Gluconate Metabolism Reveals a Novel Pathway for l-Idonic Acid Catabolism in Escherichia coli

The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of l-idonic acid in which d-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnR operon, which encodes l-idonate 5-dehydrogenase, 5-keto-d-gluconate 5-reductase, an l-idonate transporter, and an l-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of l-idonate; IdnD catalyzes a reversible oxidation of l-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to form d-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of d-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, with l-idonate or 5-ketogluconate serving as the true inducer of the pathway. The l-idonate 5-dehydrogenase and 5-keto-d-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of l-idonate and d-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize l-idonate as the sole carbon and energy source, and as predicted, the ability of idnD, idnK, idnR, and edd mutants to grow on l-idonate is altered.In Escherichia coli, the Entner-Doudoroff (ED) pathway serves as a metabolic “funnel” receiving intermediates formed by catabolism of several sugar acids (17). Hexuronic acids undergo rearrangement in the inducible Ashwell pathways (1) to form 2-keto-3-deoxygluconate, which is then phosphorylated to produce 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDPG is cleaved by KDPG aldolase, encoded by eda, providing for entry of carbon into glycolysis. The other enzyme of the ED pathway is 6-phosphogluconate dehydratase, encoded by edd, which is induced only for catabolism of gluconate and also forms KDPG, the key intermediate of the ED pathway (7). Long considered to be of more significance than is readily obvious (9), the finding that eda and edd eda double mutants are unable to colonize the mouse large intestine underscores the possible ecological importance of ED metabolism (32). The implication from these colonization studies is that colonic mucus, which contains several sugar acids, may serve as an important source of nutrients for E. coli in the gut.Also participating in gluconate catabolism are several gluconate transporters and two gluconate kinases which appear, based upon their regulation, to comprise two distinct systems (2, 13). The GntI (main) system consists of gntT, gntU, and gntK, which code for high- and low-affinity gluconate transporters and a thermoresistant gluconate kinase, respectively (23–25, 33). Expression of the GntR regulon, that is, GntI together with the edd-eda operon, is negatively controlled by the gntR gene product. The GntII (subsidiary) system is comprised of a thermosensitive gluconate kinase and a gluconate transporter which function for gluconate catabolism in the absence of the GntI system (2, 11, 13, 22). It appears that the subsidiary gluconate transporter, which has an apparent K_m for gluconate of 60 μM (23), is encoded by a gene (idnT) which is adjacent to the gene encoding the thermosensitive gluconokinase (idnK) at 96.8 min.The DNA sequence of the GntII system genes, located at 4492 kb on the genome, was revealed by the E. coli Genome Project (5, 6). If the GntII system had evolved as a subsidiary pathway for gluconate catabolism, one would expect it to contain only a gluconate transporter and gluconate kinase. However, in addition to the divergent idnK and idnT genes, this region also encodes two “dehydrogenase-like” enzymes. The similarity of idnO to gno of Gluconobacter oxydans, which encodes d-gluconate:NADP 5-oxidoreductase (GNO) (15), led to the testing of ketogluconates as enzyme substrates for the two newly identified dehydrogenases. A process of deductive reasoning and biochemical experiments led to the conclusion that the GntII system in fact comprises a novel metabolic pathway for catabolism of l-idonic acid, in which gluconate is a key intermediate. Accordingly, the genes involved in l-idonate metabolism have been given the designation idn (see Table ​Table11 for gene nomenclature).

TABLE 1

Gene and enzyme nomenclature^a

Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.     

A Novel TCR Transgenic Model Reveals That Negative Selection Involves an Immediate,Bim-Dependent Pathway and a Delayed,Bim-Independent Pathway

A complete understanding of negative selection has been elusive due to the rapid apoptosis and clearance of thymocytes in vivo. We report a TCR transgenic model in which expression of the TCR during differentiation occurs only after V(D)J-like recombination. TCR expression from this transgene closely mimics expression of the endogenous TCRα locus allowing for development that is similar to wild type thymocytes. This model allowed us to characterize the phenotypic changes that occurred after TCR-mediated signaling in self-reactive thymocytes prior to their deletion in a highly physiological setting. Self-reactive thymocytes were identified as being immature, activated and CD4^loCD8^lo. These cells had upregulated markers of negative selection and were apoptotic. Elimination of Bim reduced the apoptosis of self-reactive thymocytes, but it did not rescue their differentiation and the cells remained at the immature CD4^loCD8^lo stage of development. These cells upregulate Nur77 and do not contribute to the peripheral T cell repertoire in vivo. Remarkably, development past the CD4^loCD8^lo stage was possible once the cells were removed from the negatively selecting thymic environment. In vitro development of these cells occurred despite their maintenance of high intracellular levels of Nur77. Therefore, in vivo, negatively selected Bim-deficient thymocytes are eliminated after prolonged developmental arrest via a Bim-independent pathway that is dependent on the thymic microenvironment. These data newly reveal a layering of immediate, Bim-dependent, and delayed Bim-independent pathways that both contribute to elimination of self-reactive thymocytes in vivo.     

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ?-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.     

A New 4-Nitrotoluene Degradation Pathway in a Mycobacterium Strain

Mycobacterium sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the Mycobacterium strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-m-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-m-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-m-cresol, observed during growth of the Mycobacterium strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (λ_max, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-m-cresol by meta ring cleavage.     

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the β-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2′,3′-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn²⁺ and Ni²⁺ for catalysis, whereas Zn²⁺ afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn²⁺ ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn²⁺ binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.In many environments inorganic phosphate, an essential nutrient, can fall to extremely low concentrations, forcing microorganisms to utilize other forms of phosphorus to survive. In such cases, organophosphonates can comprise a major fraction of the total phosphorus available to biological systems (e.g. 2-aminoethylphosphonate is a widespread natural product). However, cleavage of the highly stable carbon-phosphorus (CP)⁵ bond to release inorganic phosphate requires specialized enzymes. One such enzyme activity found widely in bacteria is CP-lyase (1). Cleavage of the CP bond of organophosphonates by CP-lyase yields inorganic phosphate and, remarkably, a hydrocarbon. CP-lyase is actually a multienzyme system, encoded by the phn operon (phnCDEFGHIJKLMNOP), which is induced by low concentrations of phosphate as part of the pho regulon. Gene deletion studies in Escherichia coli have shown that phnGHIJKLM are essential for catalysis of CP bond cleavage, whereas the remaining genes probably encode transport, regulatory, or accessory functions (2). Only a handful of the proteins encoded by the phn operon have been characterized to date. PhnD was shown to be a periplasmic binding protein with high affinity for organophosphonates (3); the three-dimensional structure of PhnH, one of the proteins essential for CP-lyase catalysis, was recently solved, but a function has yet to be determined (4); PhnN was shown to be an ATP-dependent kinase that provides a redundant pathway to 5-phospho-d-ribofuranosyl-α-1-diphosphate (5); and PhnO was demonstrated to be an acetyl-CoA-dependent N-acyltransferase with activity toward a wide range of aminoalkylphosphonates (6).Although the phnP gene is not essential for CP bond cleavage by cells in liquid culture (2), cell growth on solid media supplemented with methylphosphonate or phosphite as the sole phosphorus source is prevented by phnP mutations (7), suggesting a critical regulatory or accessory role for PhnP. Accordingly, phnP appears frequently in the phn operon in various species of bacteria, typically following the phnN gene (8). PhnP is predicted based on its sequence to be a member of the β-lactamase family of metal-dependent hydrolases with greatest homology to enzymes from the tRNase Z (ProDom family PD352433) and ElaC families (9), the latter erroneously annotated as composed of arylsulfatases but later determined to also belong to the tRNase Z family (10, 11). The tRNase Z enzymes are endonucleases used by prokaryotes and eukaryotes to cleave a specific phosphodiester bond near the 3′-end of pre-tRNA, yielding a 3′-end that can be coupled to an amino acid. These enzymes typically use two active site bound Zn²⁺ ions to simultaneously lower the pK_a of a nucleophilic water molecule and stabilize negative charge development on the phosphodiester linkage undergoing nucleophilic attack (12). Since it is not clear how a tRNase activity would support cell growth with an organophosphonate as a sole phosphorus source, we set out to characterize the substrate specificity and three-dimensional structure of PhnP to learn more about this critical CP-lyase enzyme.     

Genetic Analysis of Phenoxyalkanoic Acid Degradation in Sphingomonas herbicidovorans MH

Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for α-ketoglutarate-dependent dioxygenases, sdpA_MH and rdpA_MH, were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA_I/II), two for dienelactone hydrolases (dccD_I/II), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA_MH was 99% identical to rdpA_MC1, an (R)-dichlorprop/α-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA_I and DccA_II did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria.     

Large-Scale Arrayed Analysis of Protein Degradation Reveals Cellular Targets for HIV-1 Vpu

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para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable ¹³C₆-isotope of pABA (p- amino[aromatic-¹³C₆]benzoic acid ([¹³C₆]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[¹³C₆]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [¹³C₆]pABA or [¹³C₆]4HB generate both ¹³C₆-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product ¹³C₆-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ₆. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ₆ quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.     

Genetic Evidence for a Defective Xylan Degradation Pathway in Lactococcus lactis

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974–3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a β-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.     

Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.