Kazutake Hirooka

Regulation of fatty acid metabolism in bacteria

In Escherichia coli, the main player in transcription regulation of fatty acid metabolism is the FadR protein, which is involved in negative regulation of fatty acid degradation and in positive and negative regulation of the cellular processes related to it, as well as in positive regulation of the biosynthesis of unsaturated fatty acids in a concerted manner with negative regulation of FabR. On the other hand, Bacillus subtilis possesses two global transcriptional regulators, FadR (YsiA) and FapR. B. subtilis FadR represses fatty acid degradation, whereas FapR represses almost all the processes in the biosynthesis of saturated fatty acids and phospholipids. Furthermore, Streptococcus pneumoniae FabT represses the genes of fatty acid biosynthesis that are clustered in its genome. Long‐chain acyl‐CoAs appear to be metabolic signals for fatty acid degradation by bacteria in general, and antagonize the FadR protein from either E. coli or B. subtilis. However, malonyl‐CoA is a metabolic signal for fatty acid and phospholipid biosynthesis by Gram‐positive low‐GC bacteria, and it antagonizes FapR. These would be the primary aspects for understanding the elaborate and complex regulation of fatty acid metabolism in bacteria to maintain membrane lipid homeostasis.

Conversion of Product Specificity of Archaebacterial Geranylgeranyl-diphosphate Synthase IDENTIFICATION OF ESSENTIAL AMINO ACID RESIDUES FOR CHAIN LENGTH DETERMINATION OF PRENYLTRANSFERASE REACTION

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH3, which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from ~1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.

Elaborate transcription regulation of the Bacillus subtilis ilv‐leu operon involved in the biosynthesis of branched‐chain amino acids through global regulators of CcpA, CodY and TnrA

The Bacillus subtilis ilv‐leu operon involved in the biosynthesis of branched‐chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis‐elements located upstream of the ilv‐leu promoter. This operon is known to be under CcpA‐dependent positive regulation. We have currently identified a catabolite‐responsive element (cre) for this positive regulation (bases −96 to −82; +1 is the ilv‐leu transcription initiation base) by means of DNase I‐footprinting in vitro, and deletion and base‐substitution analyses of cre. Under nitrogen‐rich growth conditions in glucose‐minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv‐leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA‐dependent positive regulation did not overcome the CodY‐dependent negative regulation. Furthermore, under nitrogen‐limited conditions in glucose‐minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA‐dependent positive regulation occurred without the TnrA‐dependent negative regulation. However, the TnrA‐dependent negative regulation did not occur without the CcpA‐dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA‐dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv‐leu operon in overall metabolic regulation in this organism is discussed.

Conversion from Archaeal Geranylgeranyl Diphosphate Synthase to Farnesyl Diphosphate Synthase TWO AMINO ACIDS BEFORE THE FIRST ASPARTATE-RICH MOTIF SOLELY DETERMINE EUKARYOTIC FARNESYL DIPHOSPHATE SYNTHASE ACTIVITY

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways.

A pathway where polyprenyl diphosphate elongates in prenyltransferase. Insight into a common mechanism of chain length determination of prenyltransferases

Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase. The side chains of these amino acids are situated on the same side of an α-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996)J. Biol. Chem. 271, 18831–18837). A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40. A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40compound with C35 and C45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.

Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation.

The organization and function of the Bacillus subtilis YsiA regulon involved in fatty acid degradation were investigated. Northern and primer extension analyses indicated that this regulon comprises five operons, i.e. lcfA-ysiA-B-etfB-A, ykuF-G, yhfL, yusM-L-K-J, and ywjF-acdA-rpoE. YusJ and AcdA, YsiB and YusL, and YusK presumably encode acyl-CoA dehydrogenases, 3-hydroxyl-CoA dehydrogenase/enoyl-CoA hydratase complexes, and acetyl-CoA C-acyltransferase, respectively, which are directly involved in the fatty acid β-oxidation cycle. In addition, LcfA and YhfL are likely to encode long chain acyl-CoA ligases. On gel retardation and footprinting analyses involving the purified YsiA protein, we identified cis-sequences for YsiA binding (YsiA boxes) in the promoter regions upstream of ysiA, ykuF, yusL, yhfL, and ywjF, the equilibrium dissociation constants (Kd) for YsiA binding being 20, 21, 37, 43, and 65 nm, respectively. YsiA binding was specifically inhibited by long chain acyl-CoAs with 14–20 carbon atoms, acyl-CoAs with 18 carbon atoms being more effective; out of long chain acyl-CoAs tested, monounsaturated oleoyl-CoA, and branched chain 12-metyltetradecanoyl-CoA were most effective. These in vitro findings were supported by the in vivo observation that the knock-out of acyl-CoA dehydrogenation through yusJ, etfA, or etfB disruption resulted in YsiA inactivation, probably because of the accumulation of long chain acyl-CoAs in the cells. Furthermore, the disruption of yusL, yusK, yusJ, etfA, etfB, or ykuG affected the utilization of palmitic acid, a representative long chain fatty acid. Based on this work, ysiA, ysiB, ykuF, ykuG, yhfL, yusM, yusL, yusK, yusJ, and ywjF can be renamed fadR, fadB, fadH, fadG, lcfB, fadM, fadN, fadA, fadE, and fadF.

Enhancement of Glutamine Utilization in Bacillus subtilis through the GlnK-GlnL Two-Component Regulatory System

During DNA microarray analysis, we discovered that the GlnK-GlnL (formerly YcbA-YcbB) two-component system positively regulates the expression of the glsA-glnT (formerly ybgJ-ybgH) operon in response to glutamine in the culture medium on Northern analysis. As a result of gel retardation and DNase I footprinting analyses, we found that the GlnL protein interacts with a region (bases -13 to -56; +1 is the transcription initiation base determined on primer extension analysis of glsA-glnT) in which a direct repeat, TTTTGTN4TTTTGT, is present. Furthermore, the glsA and glnT genes were biochemically verified to encode glutaminase and glutamine transporter, respectively.

Negative Transcriptional Regulation of the ilv-leu Operon for Biosynthesis of Branched-Chain Amino Acids through the Bacillus subtilis Global Regulator TnrA

The Bacillus subtilis ilv-leu operon is involved in the synthesis of branched-chain amino acids (valine, isoleucine, and leucine). The two- to threefold repression of expression of the ilv-leu operon during logarithmic-phase growth under nitrogen-limited conditions, which was originally detected by a DNA microarray analysis to compare the transcriptomes from the wild-type and tnrA mutant strains, was confirmed by lacZ fusion and Northern experiments. A genome-wide TnrA box search revealed a candidate box approximately 200 bp upstream of the transcription initiation base of the ilv-leu operon, the TnrA binding to which was verified by gel retardation and DNase I footprinting analyses. Deletion and base substitution of the TnrA box sequence affected the ilv-leu promoter activity in vivo, implying that TnrA bound to the box might be able to inhibit the promoter activity, possibly through DNA bending. The negative control of the expression of the ilv-leu operon by TnrA, which is considered to represent rather fine-tuning (two- to threefold), is a novel regulatory link between nitrogen and amino acid metabolism.

Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme.

A gene coding for an esterase (SshEstI, 915 bp in length) of the thermoacidophilic archaeon Sulfolobus shibatae DSM5389 was cloned, sequenced, and overexpressed in Escherichia coli JM109 cells as a soluble, catalytically active protein. The deduced amino acid sequence of SshEstI was consistent with a protein containing 305 amino acid residues with a molecular mass of 33 kDa. Sequence comparison studies indicated that SshEstI could be a member of the hormone-sensitive lipase family, in that it had the highest sequence similarity to esterases from Sulfolobus solfataricus (90% identity) and Archaeoglobus fulgidus (42%) and a lipase from Pseudomonas sp. B11-1 (38%). The recombinant enzyme was highly thermostable and retained more than 70% of its initial activity after incubation at 90 degrees C and pH 7.0 for 30 min. The recombinant enzyme catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with C2-C16 acyl chains but not the hydrolysis of triacylglycerides such as tributyrin and triolein. The enzymatic hydrolysis of p-NP acetate proceeded in a linear manner with time, whereas that of p-NP esters with acyl chains of C5 or longer showed a biphasic profile, where a rapid release of p-nitrophenol ( approximately 3 min) was followed by a slow, sustained release. These non-linear kinetics may be explained in terms of a very slow, presteady-state burst phenomenon of p-nitrophenol release or a hysteretic behavior of SshEstI with these substrates.

Dual Regulation of the Bacillus subtilis Regulon Comprising the lmrAB and yxaGH Operons and yxaF Gene by Two Transcriptional Repressors, LmrA and YxaF, in Response to Flavonoids

Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter beta-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.