[Show abstract][Hide abstract] ABSTRACT: Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.
[Show abstract][Hide abstract] ABSTRACT: Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.
[Show abstract][Hide abstract] ABSTRACT: Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis-the paradigm of mesophilic hydrocarbonoclastic bacteria-O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
[Show abstract][Hide abstract] ABSTRACT: The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity and activities. Herein, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46' N, 2°59' W; 655 m altitude). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from the Lake Arreo suggests that its sediment contain moderately halophilic and cold-adapted Proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origin. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics as they exhibit maximal activity at alkaline pH (8.0-8.5), 16-40°C and are stimulated (1.5-2.2 times) by chloride ions (0.1-1.2 M) reflecting an adaption to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e. production of enantiomers and sugar esters) because these enzymes are salt-tolerant and are active at low temperatures and against a broad range of substrates. As example, the ability of a single protein to hydrolyze at similar extent triacylglycerols, (non) halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones and chiral epoxides was demonstrated.
Applied and Environmental Microbiology 06/2013; 79(12):3553-3562. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: General features of reported esterases/lipases isolated from metagenomic resources (Table S1); list of primers used in the study (Table S2); percentage of identity between Arreo enzymes, determined by Matcher (EMBOSS package) (Table S3); half-saturation (Michaelis) coefficient (Km), catalytic rate constant (kcat), and catalytic efficiency (kcat/Km) values for the wild-type α/β hydrolases from Lake Arreo (Table S4); location map, vegetation and use of drainage basin map, and bathymetric map of Lake Arreo (Fig. S1); Lake Arreo proteins, as overexpressed in the active form in E. coli at 16ºC (Fig. S2); chemical structures of substrates applied for activity profiling of esterase/lipase preparations used in the present study (Fig. S3); structural models of esterases/lipases from the α/β hydrolase family characterized in this work (Fig. S4); nucleotide and amino acid sequences of esterases/lipases from Lake Arreo (“Annex”); supplemental text: GOHTAM and BLAST analyses.
[Show abstract][Hide abstract] ABSTRACT: The human HD domain protein SAMHD1 is implicated in the Aicardi-Goutières autoimmune syndrome and in the restriction of HIV-1 replication in myeloid cell. Recently, this protein has been shown to possess dNTP triphosphatase activity, which is proposed to inhibit HIV-1 replication and the autoimmune response by hydrolysing cellular dNTPs. Here we show that the purified full-length human SAMHD1 also possesses metal-dependent 3' to 5' exonuclease activity against single stranded DNAs and RNAs in vitro. In double stranded substrates, this protein preferentially cleaved 3'-overhangs and RNA in the blunt-ended RNA/DNA duplexes. The full-length SAMHD1 also exhibited strong DNA and RNA binding to substrates with complex secondary structures. Both nuclease and dNTP triphosphatase activities of SAMHD1 are associated with its HD domain, but the SAM domain is required for maximal activity and nucleic acid binding. Nuclease activity of SAMHD1 could represent an additional mechanism contributing to HIV-1 restriction and suppression of the autoimmune response through a direct cleavage of viral and endogenous nucleic acids. In addition, we demonstrated the presence of dGTP triphosphohydrolase and nuclease activities in several microbial HD domain proteins suggesting that these proteins might contribute to anti-viral defense in prokaryotes.
Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The uncharacterized α/β-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5-30°C with maximal activity at 15-20°C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
[Show abstract][Hide abstract] ABSTRACT: The hotdog fold is one of the basic protein folds widely present in bacteria, archaea and eukaryotes. Many of these proteins exhibit thioesterase activity against fatty acyl-CoAs and play important roles in lipid metabolism, cellular signalling and degradation of xenobiotics. The genome of the opportunistic pathogen Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (at 1.70 and 1.75 Å for PA5202 and PA2801 respectively) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long α-helix (Asp(57) in PA5202 and Glu(35) in PA2801). Alanine residue replacement mutagenesis of PA5202 identified four residues (Asn(42), Arg(43), Asp(57) and Thr(76)) that are critical for its activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis, suggesting a functional link between PA5202 activity and pyocyanin production. Thus the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.
[Show abstract][Hide abstract] ABSTRACT: The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid
substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate
dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid
dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from
Pseudomonas aeruginosa catalyzes an NAD+-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å
resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The
PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the
predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD+ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near
Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including
the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity
and activity of β-hydroxyacid dehydrogenases.
Journal of Biological Chemistry 01/2012; 287(3):1874-1883. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD(+)-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2-2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD(+) complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.
Journal of Biological Chemistry 11/2011; 287(3):1874-83. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 Å resolution (1 Å=0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,β-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.
[Show abstract][Hide abstract] ABSTRACT: Cyclic diguanylate (or bis-(3'-5') cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 Å) and in complex with c-di-GMP (2.35 A), and unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.
Journal of Molecular Biology 09/2010; 402(3):524-38. · 3.91 Impact Factor