Structural insights into vesicle amine transport-1 (VAT-1) as a member of the NADPH-dependent quinone oxidoreductase family

Vesicle amine transport protein-1 (VAT-1) has been implicated in the regulation of vesicular transport, mitochondrial fusion, phospholipid transport and cell migration, and is a potential target of anticancer drugs. Little is known about the molecular function of VAT-1. The amino acid sequence indicates that VAT-1 belongs to the quinone oxidoreductase subfamily, suggesting that VAT-1 may possess enzymatic activity in unknown redox processes. To clarify the molecular function of VAT-1, we determined the three-dimensional structure of human VAT-1 in the free state at 2.3 Å resolution and found that VAT-1 forms a dimer with the conserved NADPH-binding cleft on each protomer. We also determined the structure of VAT-1 in the NADP-bound state at 2.6 Å resolution and found that NADP binds the binding cleft to create a putative active site with the nicotine ring. Substrate screening suggested that VAT-1 possesses oxidoreductase activity against quinones such as 1,2-naphthoquinone and 9,10-phenanthrenequinone.

Exo-β-N-acetylmuramidase NamZ of Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases

ABSTRACTEndo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that potentially lyse bacterial cells. They catalyze an endo-lytic cleavage of the peptidoglycan, the structural component of the bacterial cell wall; i.e. they hydrolyze glycosidic N-acetylmuramic acid (MurNAc)-β-1,4-N-acetylglucosamine (GlcNAc)-bonds within the heteroglycan backbone of peptidoglycan. In contrast, little is known about exo-β-N-acetylmuramidases, catalyzing an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We identified ybbC of B. subtilis, renamed namZ, as encoding the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. NamZ is a member of the DUF1343 protein family of unknown function and shows no significant sequence identity with known glycosidases. A structural model of NamZ revealed a putative active site located in a cleft within the interface of two subdomains, one of which constituting a Rossmann-fold-like domain, unusual for glycosidases. On this basis, we propose that NamZ represents the founding member of a novel family of peptidoglycan hexosaminidases, which is mainly present in the phylum Bacteroidetes and, less frequently, within Firmicutes (Bacilli, Clostridia), Actinobacteria and Gammaproteobacteria.

The histone H3-H4 tetramer is a copper reductase enzyme

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3′ dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae. The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.

Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl cis/trans isomerases (PPIs). The Escherichia coli cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6ppi strains could be constructed only on minimal medium in the temperature range of 30–37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the ppiC gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC’s PPIase activity was shown to be required for interaction with AhpC.

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

SUMMARYMycobacterium tuberculosis causes tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides – arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 Å resolution structure of M. abscessus AftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 Å structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed in M. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

Crystal Structures of the Putative Isocitrate Dehydrogenase from Sulfolobus tokodaii Strain 7 in the Apo and NADP+-Bound Forms

Isocitrate dehydrogenase is a catabolic enzyme that acts during the third step of the tricarboxylic acid cycle. The hypothetical protein ST2166 from the archaeon Sulfolobus tokodaii was isolated and crystallized. It shares high primary structure homology with prokaryotic NADP+-dependent IDHs, suggesting that these enzymes share a common enzymatic mechanism. The crystal structure of ST2166 was determined at 2.0 Å resolution in the apo form, and then the structure of the crystal soaked with NADP+ was also determined at 2.4 Å resolution, which contained NADP+ bound at the putative active site. Comparisons between the structures of apo and NADP+-bound forms and NADP-IDHs from other prokaryotes suggest that prokaryotic NADP-IDHs recognize their cofactors using conserved Lys335, Tyr336, and Arg386 in ST2166 at the opening cleft before the domain closure.

The Histone H3-H4 Tetramer is a Copper Reductase Enzyme

Ancestral histones were present in organisms with small genomes, no nucleus, and little evidence for epigenetic regulation, suggesting histones may have additional older functions. We report that the histone H3-H4 tetramer is an enzyme that catalyzes the reduction of Cu2+ to Cu1+ when assembled in vitro from recombinant histones. Mutations of residues in the putative active site at the interface of the apposing H3 proteins alter the enzymatic activity and cellular processes such as Sod1 function or mitochondrial respiration that depend on availability of reduced copper. These effects are not due to altered gene expression or copper abundance but are consistent with decreased levels of cuprous ions. We propose that the H3-H4 tetramer is an oxidoreductase that provides biousable copper for cellular and mitochondrial chemistry. As the emergence of eukaryotes coincided with the Great Oxidation Event and decreased biousability of metals, the histone enzymatic function may have facilitated eukaryogenesis.

Expression, crystallization and structure elucidation of γ-terpinene synthase fromThymus vulgaris

The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil fromThymussp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase fromT. vulgaris(TvTPS), theThymusspecies that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purifiedTvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site ofTvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase fromSalviasp. and to (4S)-limonene synthase fromMentha spicata.

Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393

ABSTRACTXylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacteriumBacteroides intestinalisDSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. Thexyn8Agene encodes an endoxylanase (Xyn8A), andrex8Aencodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3through X6to a mixture of xylose (X1) and X2. Moreover,rex8Ais located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X2to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among theBacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.