scholarly journals Identification of Enzymes Homologous to Isocitrate Dehydrogenase That Are Involved in Coenzyme B and Leucine Biosynthesis in Methanoarchaea

2000 ◽  
Vol 182 (17) ◽  
pp. 5013-5016 ◽  
Author(s):  
David M. Howell ◽  
Marion Graupner ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Isocitrate Dehydrogenase ◽  
Protein Product ◽  
Oxidative Decarboxylation ◽  
Gene Products ◽  
Methanococcus Jannaschii ◽  
Leucine Biosynthesis ◽  
Isopropylmalate Dehydrogenase ◽  
Coenzyme B

ABSTRACT Two putative Methanococcus jannaschii isocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation of dl-threo-3-isopropylmalic acid, threo-isocitrate, erythro-isocitrate, and homologs of threo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate). These intermediates included (−)-threo-isohomocitrate [(−)-threo-1-hydroxy-1,2,4-butanetricarboxylic acid], (−)-threo-iso(homo)2citrate [(−)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid], and (−)-threo-iso(homo)3citrate [(−)-threo-1-hydroxy-1,2,6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be α-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer ofdl-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100°C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80°C for 10 min.

scholarly journals Identification of the Gene Encoding Sulfopyruvate Decarboxylase, an Enzyme Involved in Biosynthesis of Coenzyme M

2000 ◽  
Vol 182 (17) ◽  
pp. 4862-4867 ◽  
Author(s):  
Marion Graupner ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Second Step ◽  
Gene Products ◽  
Methanococcus Jannaschii ◽  
Coenzyme M ◽  
Gene Encoding ◽  
Fourth Step

ABSTRACT The products of two adjacent genes in the chromosome ofMethanococcus jannaschii are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis inStreptomyces wedmorensis. These two M. jannaschii genes were recombinantly expressed inEscherichia coli, and their gene products were tested for the ability to catalyze the decarboxylation of a series of α-ketoacids. Both subunits are required to form an α6β6 dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde. This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism. The M. jannaschiisulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite. The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.

scholarly journals E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA.

1985 ◽  
Vol 5 (10) ◽  
pp. 2653-2661 ◽  
Author(s):  
B Ferguson ◽  
B Krippl ◽  
O Andrisani ◽  
N Jones ◽  
H Westphal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Biochemical Properties ◽  
Protein Product ◽  
Gene Products ◽  
E Coli ◽  
Adenovirus E1a ◽  
Myc Gene ◽  
E1a Gene ◽  
Transcription Activators

We previously purified and characterized functionally the Escherichia coli-expressed product of the human subgroup C adenovirus E1A 13S mRNA (B. Ferguson, N. Jones, J. Richter, and M. Rosenberg, Science 224:1343-1346, 1984; B. Krippl, B. Ferguson, M. Rosenberg, and H. Westphal, Proc. Natl. Acad. Sci. USA 81:6988-6992, 1984). We have now expressed in E. coli and purified the protein product encoded by the human subgroup C adenovirus E1A 12S mRNA and have compared the functional properties of this protein with those of the E1A 13S mRNA product. Using microinjection techniques to introduce these proteins into mammalian cells, we found that the E1A 12S mRNA product, like the 13S mRNA product, localized rapidly to the cell nucleus and induced adenovirus gene expression. Although both E1A gene products localized to the nucleus and stimulated adenovirus gene transcription, these proteins did not directly bind to DNA under conditions in which a known DNA-binding protein, the human c-myc gene product, bound DNA efficiently. Thus, the E1A and myc gene products, which have been related both structurally and functionally, exhibit distinctly different biochemical properties.

scholarly journals Enzymology and Evolution of the Pyruvate Pathway to 2-Oxobutyrate in Methanocaldococcus jannaschii

2007 ◽  
Vol 189 (12) ◽  
pp. 4391-4400 ◽  
Author(s):  
Randy M. Drevland ◽  
Abdul Waheed ◽  
David E. Graham
Keyword(s):  
Functional Divergence ◽  
Small Subunit ◽  
Isomerization Reaction ◽  
Oxidative Decarboxylation ◽  
Methanocaldococcus Jannaschii ◽  
Iron Sulfur Center ◽  
Gene Recruitment ◽  
Or Gene ◽  
Isopropylmalate Dehydrogenase ◽  
Coenzyme B

ABSTRACT The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of β-isopropylmalate, β-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.

E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA

1985 ◽  
Vol 5 (10) ◽  
pp. 2653-2661
Author(s):  
B Ferguson ◽  
B Krippl ◽  
O Andrisani ◽  
N Jones ◽  
H Westphal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Biochemical Properties ◽  
Protein Product ◽  
Gene Products ◽  
E Coli ◽  
Adenovirus E1a ◽  
Myc Gene ◽  
E1a Gene ◽  
Transcription Activators

We previously purified and characterized functionally the Escherichia coli-expressed product of the human subgroup C adenovirus E1A 13S mRNA (B. Ferguson, N. Jones, J. Richter, and M. Rosenberg, Science 224:1343-1346, 1984; B. Krippl, B. Ferguson, M. Rosenberg, and H. Westphal, Proc. Natl. Acad. Sci. USA 81:6988-6992, 1984). We have now expressed in E. coli and purified the protein product encoded by the human subgroup C adenovirus E1A 12S mRNA and have compared the functional properties of this protein with those of the E1A 13S mRNA product. Using microinjection techniques to introduce these proteins into mammalian cells, we found that the E1A 12S mRNA product, like the 13S mRNA product, localized rapidly to the cell nucleus and induced adenovirus gene expression. Although both E1A gene products localized to the nucleus and stimulated adenovirus gene transcription, these proteins did not directly bind to DNA under conditions in which a known DNA-binding protein, the human c-myc gene product, bound DNA efficiently. Thus, the E1A and myc gene products, which have been related both structurally and functionally, exhibit distinctly different biochemical properties.

scholarly journals ( R )-Citramalate Synthase in Methanogenic Archaea

1999 ◽  
Vol 181 (1) ◽  
pp. 331-333 ◽  
Author(s):  
David M. Howell ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Coenzyme A ◽  
Methanogenic Archaea ◽  
Protein Product ◽  
Methanococcus Jannaschii ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The Methanococcus jannaschii gene MJ1392 was cloned, and its protein product was hyperexpressed in Escherichia coli. The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (R)-citramalate. Thus, this gene (cimA) encodes an (R)-citramalate synthase (CimA). This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine.

Characterization of 3-isopropylmalate dehydrogenase from extremely halophilic archaeon Haloarcula japonica

2021 ◽  
Author(s):  
Shintaro Nagaoka ◽  
Noriko Sugiyama ◽  
Rie Yatsunami ◽  
Satoshi Nakamura
Keyword(s):  
Analytical Ultracentrifugation ◽  
Maximum Activity ◽  
Oxidative Decarboxylation ◽  
Halophilic Archaeon ◽  
Leucine Biosynthesis ◽  
First Report ◽  
Haloarcula Japonica ◽  
Extremely Halophilic Archaeon ◽  
Isopropylmalate Dehydrogenase

Abstract 3-Isopropylmalate dehydrogenase (IPMDH) catalyzes oxidative decarboxylation of (2R, 3S)-3-isopropylmalate to 2-oxoisocaproate in leucine biosynthesis. In this study, recombinant IPMDH (HjIPMDH) from an extremely halophilic archaeon, Haloarcula japonica TR-1, was characterized. Activity of HjIPMDH increased as KCl concentration increased, and the maximum activity was observed at 3.0 M KCl. Analytical ultracentrifugation revealed that HjIPMDH formed a homotetramer at high KCl concentrations, and it dissociated to a monomer at low KCl concentrations. Additionally, HjIPMDH was thermally stabilized by higher KCl concentrations. This is the first report on haloarchaeal IPMDH.

scholarly journals New Class of IMP Cyclohydrolases in Methanococcus jannaschii

2002 ◽  
Vol 184 (5) ◽  
pp. 1471-1473 ◽  
Author(s):  
Marion Graupner ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Catalytic Properties ◽  
Sequence Similarity ◽  
Genetic Locus ◽  
Protein Product ◽  
Methanococcus Jannaschii ◽  
New Class

ABSTRACT The enzyme responsible for observed IMP cyclohydrolase activity in Methanococcus jannaschii was purified and sequenced: its genetic locus was found to correspond to gene MJ0626. The MJ0626 gene was cloned, and its protein product was expressed in Escherichia coli and shown to catalyze the cyclization of 5-formylamidoimidazole-4-carboxamide ribonucleotide to IMP. The enzyme has no sequence similarity to known enzymes, and its catalytic properties appear distinct from any characterized IMP cyclohydrolase. The purO gene for the enzyme is currently found only in the domain Archaea.

scholarly journals S-Adenosylmethionine Decarboxylase from the Archaeon Methanococcus jannaschii: Identification of a Novel Family of Pyruvoyl Enzymes

2000 ◽  
Vol 182 (23) ◽  
pp. 6667-6672 ◽  
Author(s):  
Alexander D. Kim ◽  
David E. Graham ◽  
Steven H. Seeholzer ◽  
George D. Markham
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Complete Genome Sequence ◽  
Methanococcus Jannaschii ◽  
Phylogenetic Distribution ◽  
Gene Encoding ◽  
E Coli ◽  
Adenosylmethionine Decarboxylase ◽  
High Concentrations

ABSTRACT Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems. S-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and Escherichia coli. Despite spermidine concentrations in the Methanococcales that are several times higher than in E. coli, no AdoMet decarboxylase gene was recognized in the complete genome sequence ofMethanococcus jannaschii. The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the E. coli speD gene (less than 11% identity). The M. jannaschii enzyme has been expressed inE. coli and purified to homogeneity. Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (αβ)2 complex. The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases. The sequence of theM. jannaschii enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria. The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage. Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes.

scholarly journals Protein complex formation in methionine chain-elongation and leucine biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Li-Qun Chen ◽  
Shweta Chhajed ◽  
Tong Zhang ◽  
Joseph M. Collins ◽  
Qiuying Pang ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Complete Characterization ◽  
Bimolecular Fluorescence Complementation ◽  
Chain Elongation ◽  
Substrate Channeling ◽  
Leucine Biosynthesis ◽  
Protein Complex Formation ◽  
Genes Encoding ◽  
Isopropylmalate Dehydrogenase

AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

Sign in / Sign up

Export Citation Format

Share Document