Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds.
(9/43)
The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 A resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzyme's reaction mechanism. (+info)
Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae.
(10/43)
Mutations in RRP6 result in the accumulation of aberrant polyadenylated transcripts from small nucleolar RNA genes. We exploited this observation to search for novel noncoding RNA genes in the yeast genome. When RNA from rrp6Delta yeast is compared with wild-type on whole-genome microarrays, numerous intergenic loci exhibit an increased mutant/wild type signal ratio. Among these loci, we found one encoding a new C/D box small nucleolar RNA, as well as a surprising number that gave rise to heterogeneous Trf4p-polyadenylated RNAs with lengths of approximately 250-500 nt. This class of RNAs is not easily detected in wild-type cells and appears associated with promoters. Fine mapping of several such transcripts shows they originate near known promoter elements but do not usually extend far enough to act as mRNAs, and may regulate the transcription of downstream mRNAs. Rather than being uninformative transcriptional "noise," we hypothesize that these transcripts reflect important features of RNA polymerase activity at the promoter. This activity is normally undetectable in wild-type cells because the transcripts are somehow distinguished from true mRNAs and are degraded in an Rrp6p-dependent fashion in the nucleus. (+info)
Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p.
(11/43)
Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand. (+info)
Kinetic and chemical mechanism of alpha-isopropylmalate synthase from Mycobacterium tuberculosis.
(12/43)
Mycobacterium tuberculosis alpha-isopropylmalate synthase (MtIPMS) catalyzes the condensation of acetyl-coenzyme A (AcCoA) with alpha-ketoisovalerate (alpha-KIV) and the subsequent hydrolysis of alpha-isopropylmalyl-CoA to generate the products CoA and alpha-isopropylmalate (alpha-IPM). This is the first committed step in l-leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and (1)H-NMR spectroscopy. The alpha-keto acid specificity of the enzyme is narrow, and the acyl-CoA specificity is absolute for AcCoA. In the absence of alpha-KIV, MtIPMS does not enolize the alpha protons of AcCoA but slowly hydrolyzes acyl-CoA analogues. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random bi-bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pK(a) values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of alpha-isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate-limiting, although, with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of alpha-isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. On the basis of these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed. (+info)
Properties of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae.
(13/43)
We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does. (+info)
Inferring direct regulatory targets from expression and genome location analyses: a comparison of transcription factor deletion and overexpression.
(14/43)
BACKGROUND: Effects on gene expression due to environmental or genetic changes can be easily measured using microarrays. However, indirect effects on expression can be substantial. The indirect effects of a perturbation need to be distinguished from the direct effects if we are to understand the structure and behavior of regulatory networks. RESULTS: The most direct way to perturb a transcriptional network is to alter transcription factor activity. Here, for the first time, we compare expression changes and genomic binding in a simple regulon under conditions of both low and high transcription factor activity. Specifically, we assessed the effects on expression and binding due to deletion of the yeast LEU3 transcription factor gene and effects due to elevation of Leu3 activity. Leu3 activity was elevated through overexpression and the introduction of a mutation that renders the protein constitutively active. Genes that are bound and/or regulated by Leu3 under one or both conditions were characterized in terms of their functional annotations and their predicted potential to be bound by Leu3. We also assessed the evolutionary conservation of the predicted binding potential using a novel alignment-independent method. Both perturbations yield genes that are likely to be direct targets of Leu3, including most of the classically defined targets. Additional direct targets are identified by each of the methods. However, experimental and computational criteria suggest that most genes whose expression is affected by the Leu3 genotype are unlikely to be regulated by binding of the protein. CONCLUSION: Most genes that are differentially expressed by Leu3 are not direct targets despite the exceptional simplicity of the regulon, and the unusually direct nature of the perturbations investigated. These conclusions are reached through computational analyses that support and extend chromatin immunoprecipitation data on the identities of direct targets. These results have implications for the interpretation of expression experiments, especially in cases for which chromatin immunoprecipitation data are unavailable, incomplete, or ambiguous. (+info)
Two Arabidopsis genes (IPMS1 and IPMS2) encode isopropylmalate synthase, the branchpoint step in the biosynthesis of leucine.
(15/43)
Heterologous expression of the Arabidopsis (Arabidopsis thaliana) IPMS1 (At1g18500) and IPMS2 (At1g74040) cDNAs in Escherichia coli yields isopropylmalate synthases (IPMSs; EC 2.3.3.13). These enzymes catalyze the first dedicated step in leucine (Leu) biosynthesis, an aldol-type condensation of acetyl-coenzyme A (CoA) and 2-oxoisovalerate yielding isopropylmalate. Most biochemical properties of IPMS1 and IPMS2 are similar: broad pH optimum around pH 8.5, Mg2+ as cofactor, feedback inhibition by Leu, Km for 2-oxoisovalerate of approximately 300 microM, and a Vmax of approximately 2 x 10(3) micromol min(-1) g(-1). However, IPMS1 and IPMS2 differ in their Km for acetyl-CoA (45 microM and 16 microM, respectively) and apparent quaternary structure (dimer and tetramer, respectively). A knockout insertion mutant for IPMS1 showed an increase in valine content but no changes in Leu content; two insertion mutants for IPMS2 did not show any changes in soluble amino acid content. Apparently, in planta each gene can adequately compensate for the absence of the other, consistent with available microarray and reverse transcription-polymerase chain reaction data that show that both genes are expressed in all organs at all developmental stages. Both encoded proteins accept 2-oxo acid substrates in vitro ranging in length from glyoxylate to 2-oxohexanoate, and catalyze at a low rate the condensation of acetyl-CoA and 4-methylthio-2-oxobutyrate, i.e. a reaction involved in glucosinolate chain elongation normally catalyzed by methylthioalkylmalate synthases. The evolutionary relationship between IPMS and methylthioalkylmalate synthase enzymes is discussed in view of their amino acid sequence identity (60%) and overlap in substrate specificity. (+info)
MAM3 catalyzes the formation of all aliphatic glucosinolate chain lengths in Arabidopsis.
(16/43)
Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of omega-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation. (+info)