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

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.

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

Journal of Bacteriology ◽

10.1128/jb.182.17.5013-5016.2000 ◽

2000 ◽

Vol 182 (17) ◽

pp. 5013-5016 ◽

Cited By ~ 23

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.

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Energy Conservation in Fermentations of Anaerobic Bacteria

Frontiers in Microbiology ◽

10.3389/fmicb.2021.703525 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wolfgang Buckel

Keyword(s):

Anaerobic Bacteria ◽

Human Microbiome ◽

Oxidative Decarboxylation ◽

Free Energies ◽

Glutamate Mutase ◽

Available Energy ◽

Glycyl Radical ◽

Oxygen Sensitive ◽

Coenzyme B ◽

Radical Enzymes

Anaerobic bacteria ferment carbohydrates and amino acids to obtain energy for growth. Due to the absence of oxygen and other inorganic electron acceptors, the substrate of a fermentation has to serve as electron donor as well as acceptor, which results in low free energies as compared to that of aerobic oxidations. Until about 10 years ago, anaerobes were thought to exclusively use substrate level phosphorylation (SLP), by which only part of the available energy could be conserved. Therefore, anaerobes were regarded as unproductive and inefficient energy conservers. The discovery of electrochemical Na+ gradients generated by biotin-dependent decarboxylations or by reduction of NAD+ with ferredoxin changed this view. Reduced ferredoxin is provided by oxidative decarboxylation of 2-oxoacids and the recently discovered flavin based electron bifurcation (FBEB). In this review, the two different fermentation pathways of glutamate to ammonia, CO2, acetate, butyrate and H2 via 3-methylaspartate or via 2-hydroxyglutarate by members of the Firmicutes are discussed as prototypical examples in which all processes characteristic for fermentations occur. Though the fermentations proceed on two entirely different pathways, the maximum theoretical amount of ATP is conserved in each pathway. The occurrence of the 3-methylaspartate pathway in clostridia from soil and the 2-hydroxyglutarate pathway in the human microbiome of the large intestine is traced back to the oxygen-sensitivity of the radical enzymes. The coenzyme B12-dependent glutamate mutase in the 3-methylaspartate pathway tolerates oxygen, whereas 2-hydroxyglutaryl-CoA dehydratase is extremely oxygen-sensitive and can only survive in the gut, where the combustion of butyrate produced by the microbiome consumes the oxygen and provides a strict anaerobic environment. Examples of coenzyme B12-dependent eliminases are given, which in the gut are replaced by simpler extremely oxygen sensitive glycyl radical enzymes.

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

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab122 ◽

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.

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Biology of Giardia lamblia

Clinical Microbiology Reviews ◽

10.1128/cmr.14.3.447-475.2001 ◽

2001 ◽

Vol 14 (3) ◽

pp. 447-475 ◽

Cited By ~ 726

Author(s):

Rodney D. Adam

Keyword(s):

Giardia Lamblia ◽

Antigenic Variation ◽

Small Subunit ◽

Genome Project ◽

Surface Proteins ◽

Small Subunit Rrna ◽

Promoter Regions ◽

Separate Species ◽

A Genome ◽

Or Gene

SUMMARY Giardia lamblia is a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from other Giardia species by light or electron microscopy. The two major genotypes of G. lamblia that infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal that Giardia spp. are among the most primitive eukaryotes. G. lamblia probably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5′ and 3′ untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from one vsp gene to another have not been associated with sequence alterations or gene rearrangements. The Giardia genome project promises to greatly increase our understanding of this interesting and enigmatic organism.

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Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081693 ◽

1978 ◽

Vol 36 (2) ◽

pp. 166-167 ◽

Cited By ~ 1

Author(s):

G. Stöffler ◽

R.W. Bald ◽

J. Dieckhoff ◽

H. Eckhard ◽

R. Lührmann ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Ribosomal Subunit ◽

Spatial Arrangement ◽

Small Subunit ◽

Antibody Binding ◽

Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

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Ribosome Structural Organization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051670 ◽

1974 ◽

Vol 32 ◽

pp. 332-333

Author(s):

James A. Lake

Keyword(s):

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Small Subunit ◽

Structural Studies ◽

X Ray Diffraction ◽

Specific Antibodies ◽

X Ray ◽

E Coli ◽

Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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Depletion and Reconstitution of Active E. Coli Ribosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116826 ◽

1975 ◽

Vol 33 ◽

pp. 640-641

Author(s):

M. Boublik ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Protein Biosynthesis ◽

Large Subunit ◽

High Resolution Electron Microscopy ◽

Small Subunit ◽

Structure And Function ◽

Biologically Active ◽

Biological Macromolecules ◽

E Coli ◽

Resolution Electron ◽

And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

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