Angeli Lal Menon

Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

Microbial metalloproteomes are largely uncharacterized

Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

Carboxy-terminal processing of the large subunit of [NiFe] hydrogenases

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co‐migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C‐terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C‐terminal cleavage between His536 and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582 and Val583, in the E. coli hydrogenase‐1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

Metals in biology: defining metalloproteomes

The vital nature of metal uptake and balance in biology is evident in the highly evolved strategies to facilitate metal homeostasis in all three domains of life. Several decades of study on metals and metalloproteins have revealed numerous essential bio-metal functions. Recent advances in mass spectrometry, X-ray scattering/absorption, and proteomics have exposed a much broader usage of metals in biology than expected. Even elements such as uranium, arsenic, and lead are implicated in biological processes as part of an emerging and expansive view of bio-metals. Here we discuss opportunities and challenges for established and newer approaches to study metalloproteins with a focus on technologies that promise to rapidly expand our knowledge of metalloproteins and metal functions in biology.

Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii

The Azotobacter vinelandii [NiFe]hydrogenase-encoding structural genes were isolated from an A. vinelandii genomic cosmid library. Nucleotide (nt) sequence analysis showed that the two genes, hoxK and hoxG, which encode the small and large subunits of the enzyme, respectively, form part of an operon that contains at least one other gene. The hoxK gene encodes a polypeptide of 358 amino acids (aa) (39,209 Da). The deduced aa sequence encodes a possible 45-aa N-terminus extension, not present in the purified A. vinelandii hydrogenase small subunit, which could be a cellular targeting sequence. The hoxG gene is downstream form, and overlaps hoxK by 4 nt and encodes a 602-aa polypeptide of 66,803 Da. The hoxK and hoxG gene products display homology to aa sequences of hydrogenase small and large subunits, respectively, from other organisms. The hoxG gene lies 16 nt upstream from a third open reading frame which could encode a 27,729-Da (240-aa) hydrophobic polypeptide containing 53% nonpolar and 11% aromatic aa. The significance of this possible third gene is not known at present.

Parallel Evolution of Transcriptome Architecture during Genome Reorganization

Assembly of genes into operons is generally viewed as an important process during the continual adaptation of microbes to changing environmental challenges. However, the genome reorganization events that drive this process are also the roots of instability for existing operons. We have determined that there exists a statistically significant trend that correlates the proportion of genes encoded in operons in archaea to their phylogenetic lineage. We have further characterized how microbes deal with operon instability by mapping and comparing transcriptome architectures of four phylogenetically diverse extremophiles that span the range of operon stabilities observed across archaeal lineages: a photoheterotrophic halophile (Halobacterium salinarum NRC-1), a hydrogenotrophic methanogen (Methanococcus maripaludis S2), an acidophilic and aerobic thermophile (Sulfolobus solfataricus P2), and an anaerobic hyperthermophile (Pyrococcus furiosus DSM 3638). We demonstrate how the evolution of transcriptional elements (promoters and terminators) generates new operons, restores the coordinated regulation of translocated, inverted, and newly acquired genes, and introduces completely novel regulation for even some of the most conserved operonic genes such as those encoding subunits of the ribosome. The inverse correlation (r=-0.92) between the proportion of operons with such internally located transcriptional elements and the fraction of conserved operons in each of the four archaea reveals an unprecedented view into varying stages of operon evolution. Importantly, our integrated analysis has revealed that organisms adapted to higher growth temperatures have lower tolerance for genome reorganization events that disrupt operon structures.

[12] 2-keto acid oxidoreductases from Pyrococcus furiosus and Thennococcus litoralis

Publisher Summary In most aerobic organisms the oxidative decarboxylation of pyruvate to acetyl-CoA is catalyzed by a large NAD-dependent pyruvate dehydrogenase complex. However, the same reaction in anaerobic organisms is catalyzed by a reversible, ferredoxin-dependent, pyruvate oxidoreductase (POR). PORs have been purified from archaea, bacteria, and anaerobic eukaryotic protists. A number of organisms, mostly archaea, are known to contain at least three 2-keto acid oxidoreductases (KORs) in addition to POR, and these are able to utilize substrates other than pyruvate. The enzymes are indolepyruvate ferredoxin oxidoreductase (IOR), 2-ketoisovalerate ferredoxin oxidoreductase (VOR), and 2-ketoglutarate ferredoxin oxidoreductase (KGOR). This article describes the purification of POR, VOR, and IOR from P. furiosus and KGOR from T. litoralis and some of the molecular and catalytic properties of this enzyme family.

Novel Multiprotein Complexes Identified in the Hyperthermophilic Archaeon Pyrococcus furiosus by Non-denaturing Fractionation of the Native Proteome

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 °C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing ∼80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.