Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

Kuznetsova, Ekaterina; Nocek, B.; Brown, Greg; Makarova, Kira S.; Flick, Robert; Wolf, Yuri I.; Khusnutdinova, Anna N.; Evdokimova, E.; Jin, Ke; Tan, Kemin; Hanson, Andrew D.; Hasnain, Ghulam; Zallot, Rémi; Crécy‐Lagard, Valérie de; Babu, Mohan; Savchenko, Alexei; Joachimiak, A.; Edwards, A.M.; Koonin, Eugene V.; Yakunin, Alexander F.

doi:10.1074/jbc.m115.657916

Cited by 81 publications

(79 citation statements)

References 138 publications

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“…All three proteins showed their highest activity against ThMP; they also showed substantial activity against flavin mononucleotide and ThDP, as well as somewhat less against inorganic pyrophosphate, CTP, and dATP ( Figure 4B). The activities against ThDP, pyrophosphate, and nucleoside triphosphates, i.e., phosphoanhydride hydrolase activities, are quite unusual for HAD phosphatases, most of which act only on monophosphates (Burroughs et al, 2006;Kuznetsova et al, 2006Kuznetsova et al, , 2015. For all three enzymes, activities against the other 89 substrates tested were very low (<4% of that with ThMP) or undetectable.…”

Section: Complementation and Insertional Mutation Confirm At5g32470 Amentioning

confidence: 97%

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Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

et al. 2016

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To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiaminrequiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470. Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

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Section: Complementation and Insertional Mutation Confirm At5g32470 Amentioning

confidence: 97%

“…Genes and enzymes are known for all the steps in the plant pathway except for a deadenylation step in thiazole synthesis and for ThMP dephosphorylation (Gerdes et al, 2012). This is also basically the case for the yeast pathway (Nosaka, 2006;Begley et al, 2012;Kuznetsova et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

et al. 2016

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“…MAPKs are highly conserved Ser/Thr protein kinases that have been extensively studied for their central roles in mediating signal transduction of extracellular stimuli to the appropriate biological response (35). MAPKs are activated by dual phosphorylation of threonine and tyrosine residues in a Thr-X-Tyr motif located in their activation loops by upstream kinases (MAPK kinases, or All HAD-like domains share a common overall fold featuring a core Rossmann fold, consisting of at least two pairs of α-helices that sandwich the core five-stranded parallel β-sheet in the order '54123', with a squiggle and flap motif at the end of β1-strand (45,46). The common molecular architecture between HAD superfamily members includes four (I to IV) highly conserved sequence motifs co-localized to the active site.…”

Section: Introductionmentioning

confidence: 99%

The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways

Quaile

Stogios

Egorova

et al. 2018

Journal of Biological Chemistry

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Host colonization by Gram-negative pathogens often involves delivery of bacterial proteins called "effectors" into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins is the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase-hydrolase domain. Ceg4 could dephosphorylate a broad range of phosphotyrosine-containing peptides in vitro and attenuated activation of MAPKcontrolled pathways in both yeast and human cells. Our findings indicate that L. pneumophila's infectious program includes manipulation of phosphorylation cascades in key host pathways. The structural and functional features of the Ceg4 effector unraveled here, provide first insight into its function as a phosphotyrosine phosphatase, paving the way to further studies into L. pneumophila pathogenicity.Host colonization by Gram-negative pathogens often involves delivery of specific sets of bacterial proteins called "effectors" into the host cell by specialized secretion systems. Acting in concert, effectors secure the pathogen's survival and replication, via manipulation of host processes and the establishment of subcellular environments that favor the pathogen. Legionella phosphotyrosine phosphatase activity towards MAPKs 2 different pathogens. For example, plague and intestinal disease-causing Yersinia species encode less than ten effectors delivered by the type III secretion system (4) while the similar secretion system in Shigella spp. is involved in translocation of close to 30 effectors (5). Notably, sequence-related effectors are found in pathogens with very diverse invasion strategies suggesting that these effector families are involved in common host manipulation tactics.A causative agent of severe pneumonia in humans, the Legionella pneumophila genome encodes over 330 effector proteins, that are translocated via the Dot/Icm (defect in organelle trafficking/ intracellular multiplication), type IVB secretion system (e.g. (6-11), or see (12) for a recent review). Legionella's effectors account for more than 10% of its proteome (13) and represents the largest effector set known to the bacterial world. In their natural habitat of fresh water reservoirs Legionella spp. invade diverse amoebae by preventing formation of the phagolysosome (14) followed by modification of the newly co-opted compartment into an organelle ideal for intracellular replication of the bacteria, the Legionella containing vacuole (LCV) (15). The ability to apply the same invasion strategy to invade human alveolar macrophages raises the intriguing possibility that Legionella's effectors target host processes that are conser...

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“…The AKRs studied in this work (see Table S1 in the supplemental material) were cloned, overexpressed in E. coli, and affinity purified (see Fig. S1) as previously described (37). Site-directed mutagenesis was performed using the QuikChange sitedirected mutagenesis kit (Stratagene) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

Kim

Flick

Brunzelle

et al. 2017

Appl Environ Microbiol

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The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in k cat /K m . Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates.IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.KEYWORDS aldo-keto reductase, 1,3-butanediol, crystal structure, site-directed mutagenesis, biocatalysis, biotechnology P resently, most of the large-volume chemicals and polymer building blocks used by the chemical industry are manufactured from nonrenewable feedstocks, such as oil and natural gas. Each year, over 80 million tons of industrial chemicals are produced

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Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

Cited by 81 publications

References 138 publications

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

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