A detailed biochemical characterization of phosphopantothenate synthetase, a novel enzyme involved in coenzyme A biosynthesis in the Archaea

We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4′-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion. Here, we examined the enzymatic properties of the archaeal phosphopantothenate synthetase, which catalyzes the ATP-dependent condensation of 4-phosphopantoate and β-alanine. The activation energy of the phosphopantothenate synthetase reaction was 82.3?kJ?mol^?1. In terms of substrate specificity toward nucleoside triphosphates, the enzyme displayed a strict preference for ATP. Among several amine substrates, activity was detected with β-alanine, but not with γ-aminobutyrate, glycine nor aspartate. The phosphopantothenate synthetase reaction followed Michaelis–Menten kinetics toward β-alanine, whereas substrate inhibition was observed with 4-phosphopantoate and ATP. Feedback inhibition by CoA/acetyl-CoA and product inhibition by 4′-phosphopantothenate were not observed. By contrast, the other archaeal enzyme pantoate kinase displayed product inhibition by 4-phosphopantoate in a non-competitive manner. Based on our results, we discuss the regulation of CoA biosynthesis in the archaea.     

Crystal structure of pantoate kinase from Thermococcus kodakarensis

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4′-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.     

Biochemical characterization of pantoate kinase, a novel enzyme necessary for coenzyme a biosynthesis in the archaea

Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4'-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from T. kodakarensis. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable k(cat)/K(m) values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg(2+), respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.     

The crystal structure of a novel phosphopantothenate synthetase from the hyperthermophilic archaea, Thermococcus onnurineus NA1

Pantothenate is the essential precursor of coenzyme A (CoA), a fundamental cofactor in all aspects of metabolism. In bacteria and eukaryotes, pantothenate synthetase (PS) catalyzes the last step in the pantothenate biosynthetic pathway, and pantothenate kinase (PanK) phosphorylates pantothenate for its entry into the CoA biosynthetic pathway. However, genes encoding PS and PanK have not been identified in archaeal genomes. Recently, a comparative genomic analysis and the identification and characterization of two novel archaea-specific enzymes show that archaeal pantoate kinase (PoK) and phosphopantothenate synthetase (PPS) represent counterparts to the PS/PanK pathway in bacteria and eukaryotes. The TON1374 protein from Thermococcus onnurineus NA1 is a PPS, that shares 54% sequence identity with the first reported archaeal PPS candidate, MM2281, from Methanosarcina mazei and 91% sequence identity with TK1686, the PPS from Thermococcus kodakarensis. Here, we report the apo and ATP-complex structures of TON1374 and discuss the substrate-binding mode and reaction mechanism.     

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2‐oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk‐KPR) displayed reductase activity specific for 2‐oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH‐dependent enzymes. Tk‐KPR activity decreased dramatically in the presence of CoA and KPR activity in cell‐free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.     

Crystal structure of archaeal ketopantoate reductase complexed with coenzyme a and 2‐oxopantoate provides structural insights into feedback regulation

Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P)H‐dependent reduction of 2‐oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2‐oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2‐oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea. Proteins 2016; 84:374–382. © 2016 Wiley Periodicals, Inc.     

Pantoate Kinase and Phosphopantothenate Synthetase, Two Novel Enzymes Necessary for CoA Biosynthesis in the Archaea

Bacteria/eukaryotes share a common pathway for coenzyme A (CoA) biosynthesis. Although archaeal genomes harbor homologs for most of these enzymes, homologs of bacterial/eukaryotic pantothenate synthetase (PS) and pantothenate kinase (PanK) are missing. PS catalyzes the ATP-dependent condensation of pantoate and β-alanine to produce pantothenate, whereas PanK catalyzes the ATP-dependent phosphorylation of pantothenate to produce 4′-phosphopantothenate. When we examined the cell-free extracts of the hyperthermophilic archaeon Thermococcus kodakaraensis, PanK activity could not be detected. A search for putative kinase-encoding genes widely distributed in Archaea, but not present in bacteria/eukaryotes, led to four candidate genes. Among these genes, TK2141 encoded a protein with relatively low PanK activity. However, higher levels of activity were observed when pantothenate was replaced with pantoate. V_max values were 7-fold higher toward pantoate, indicating that TK2141 encoded a novel enzyme, pantoate kinase (PoK). A search for genes with a distribution similar to TK2141 led to the identification of TK1686. The protein product catalyzed the ATP-dependent conversion of phosphopantoate and β-alanine to produce 4′-phosphopantothenate and did not exhibit PS activity, indicating that TK1686 also encoded a novel enzyme, phosphopantothenate synthetase (PPS). Although the classic PS/PanK system performs condensation with β-alanine prior to phosphorylation, the PoK/PPS system performs condensation after phosphorylation of pantoate. Gene disruption of TK2141 and TK1686 led to CoA auxotrophy, indicating that both genes are necessary for CoA biosynthesis in T. kodakaraensis. Homologs of both genes are widely distributed among the Archaea, suggesting that the PoK/PPS system represents the pathway for 4′-phosphopantothenate biosynthesis in the Archaea.Coenzyme A (CoA)² and its derivative 4′-phosphopantetheine are essential cofactors in numerous metabolic pathways, including the tricarboxylic acid cycle, the β-oxidation pathway, and fatty acid and polyketide biosynthesis pathways. Acyl-CoA derivatives are key intermediates in energy metabolism due to their high energy thioester bonds and have been identified in all three domains of life.The mechanism of CoA biosynthesis in bacteria and eukaryotes has been well examined and involves common enzymatic conversions (1–3). CoA is synthesized from pantothenate via five enzymatic reactions; pantothenate kinase (PanK), 4′-phosphopantothenoylcysteine synthetase (PPCS), 4′-phosphopantothenoylcysteine decarboxylase (PPCDC), 4′- phosphopantetheine adenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK). Although many animals rely on exogenous pantothenate to initiate CoA biosynthesis, microorganisms and plants can synthesize pantothenate from 2-oxoisovalerate and β-alanine. This is a three-step pathway catalyzed by ketopantoate hydroxymethyltransferase (KPHMT), ketopantoate reductase, and pantothenate synthetase (PS).In contrast to the wealth of knowledge on CoA biosynthesis in bacteria and eukaryotes, the corresponding pathway in the Archaea remains unclear (4). Sequence data indicate that the bacterial PPCS and PPCDC homologs and eukaryotic PPAT homologs are found on almost all of the archaeal genomes. The archaeal PPCS and PPCDC genes are fused in many cases, and the bifunctional protein from Methanocaldococcus jannaschii has been shown to exhibit both activities (5). The PPAT homolog from Pyrococcus abyssi has also been studied and confirmed to exhibit the expected PPAT activity (6). Bacterial KPHMT and ketopantoate reductase homologs can also be found, to a lesser extent, on the archaeal genomes. They are not found in the methanogens and Thermoplasmatales, and the fact that the structural similarity among archaeal enzymes is not higher than that toward enzymes from hyperthermophilic bacteria suggests that the archaeal KPHMT and ketopantoate reductase are a result of horizontal gene transfer from bacteria (4). In addition, there are candidate genes distantly related to bacterial/eukaryotic DPCK. However, PS homologs are not found in any of the archaeal genomes, and PanK homologs are found only in a few exceptional cases. Recently, Genschel and co-workers have taken a comparative genomics approach to predict the genes corresponding to the archaeal PS and PanK genes, and have also described the identification of a structurally novel PS from Methanosarcina mazei (4, 7).In this study, we describe the identification of the enzymes responsible for the conversion of pantoate to 4′-phosphopantothenate in Thermococcus kodakaraensis. The organism is a hyperthermophilic archaeon isolated from Kodakara Island, Japan (8, 9). The complete genome sequence is available (10), and gene disruption systems have been developed (11–13). To our surprise, the conversion of pantoate to 4′-phosphopantothenate in T. kodakaraensis is not brought about by the two classic enzyme reactions catalyzed by PS and PanK, but by two novel enzyme reactions; phosphorylation of pantoate (pantoate kinase) followed by the condensation of 4-phosphopantoate and β-alanine (4′-phosphopantothenate synthetase or 4-phosphopantoate:β-alanine ligase). Homologs of these two genes are distributed on almost all of the archaeal genomes, suggesting that the Archaea utilize different chemistry in the conversion from pantoate to 4′-phosphopantothenate.     

Latitudinal differences in temperature effects on the embryonic development and hatchling phenotypes of the Asian yellow pond turtle,Mauremys mutica

The effect of incubation temperature on embryonic development and offspring traits has been widely reported for many species. However, knowledge remains limited about how such effects vary across populations. Here, we investigated whether incubation temperature (26, 28, and 30 °C) differentially affects the embryonic development of Asian yellow pond turtle (Mauremys mutica) eggs originating from low‐latitude (Guangzhou, 23°06′N) and high‐latitude (Haining, 30°19′N) populations in China. At 26 °C, the duration of incubation was shorter in the high‐latitude population than in the low‐latitude population. However, this pattern was reversed at 30 °C. As the incubation temperature increased, hatching success increased in the low‐latitude population but slightly decreased in the high‐latitude population. Hatchlings incubated at 30 °C were larger and righted themselves more rapidly than those incubated at 26 °C in the low‐latitude population. In contrast, hatchling traits were not influenced by incubation temperature in the high‐latitude population. Overall, 30 °C was a suitable developmental temperature for embryos from the low‐latitude population, whereas 26 and 28 °C were suitable for those from the high‐latitude population. This interpopulation difference in suitable developmental temperatures is consistent with the difference in the thermal environment of the two localities. Therefore, similarly to posthatching individuals, reptile embryos from different populations might have evolved diverse physiological strategies to benefit from the thermal environment in which they develop. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 35–43.     

Anabaena sp. DyP‐type peroxidase is a tetramer consisting of two asymmetric dimers

DyP‐type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure‐based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49–55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP‐type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224‐Cys224 disulfide‐linked dimers. The tetramer of wild‐type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration‐dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non‐crystallographic, 2‐fold symmetry in the asymmetric unit, two subunits forming the Cys224‐Cys224 disulfide‐linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure‐based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes. Proteins 2016; 84:31–42. © 2015 Wiley Periodicals, Inc.     

The structure of S. lividans acetoacetyl‐CoA synthetase shows a novel interaction between the C‐terminal extension and the N‐terminal domain

The adenosine monoposphate‐forming acyl‐CoA synthetase enzymes catalyze a two‐step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ～110 residue C‐terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl‐CoA synthetase (AacS) is presented that illustrates a novel aspect of this C‐terminal domain. Specifically, several acetyl‐ and acetoacetyl‐CoA synthetases contain a 30‐residue extension on the C‐terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N‐terminal domain. Proteins 2015; 83:575–581. © 2014 Wiley Periodicals, Inc.     

ATP-induced Changes in Energy Status and Membrane Integrity of Harvested Litchi Fruit and its Relation to Pathogen Resistance

Litchi is a subtropical fruit of high commercial value on the international market but the fruit deteriorates rapidly after harvest due to rot development caused by Peronophythora litchii. To investigate the role of energy metabolism during disease development on harvested litchi fruit, fruits were dipped into solutions of either 0 or 1.0 mm adenosine triphosphate (ATP) for 3 min before being inoculated with Peronophythora litchii or not. Fruit were then stored for 6 days at 25°C and 90–100% relative humidity. Significant reductions in pericarp browning and disease severity and significant delays in membrane permeability and malondialdehyde (MDA) content were found in ATP‐treated and P. litchii‐inoculated fruit. Higher ATP concentrations and adenylate energy charge (EC) were observed in ATP‐treated fruit. In addition, lower activities of phospholipase D, acid phosphatase and lipoxygenase enzymes involved in membrane lipid peroxidation and hydrolysis were recorded in ATP‐treated fruit. Thus, treatment with ATP maintained higher energy levels, inhibited activities of the membrane hydrolysis‐related enzymes, reduced membrane lipid peroxidation and helped maintain membrane integrity of the harvested litchi fruit at the early stage of storage, which could account for the inhibition of disease development of P. litchii‐inoculated fruit.     

Agrobacterium tumefaciens mediated transient expression of plant cell wall‐degrading enzymes in detached sunflower leaves

For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S‐driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA‐Based Overexpression Vector) systems. For 6‐day leaf incubations, codon‐optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two‐phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6‐fold. Production also increased up to 4.2‐fold when the leaf incubation temperature was elevated above the typical temperature, 20°C, to 30°C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co‐expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall‐degrading enzymes in sunflower. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:905–915, 2014     

Purification and characterization of intracellular and extracellular α‐glucosidases from Geobacillus toebii strain E134

Two different α‐glucosidase‐producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo‐α‐1,4‐glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α‐glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 K_m values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α‐1,3 and α‐1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α‐glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright © 2011 John Wiley & Sons, Ltd.     

High diversity of ammonia‐oxidizing archaea in permanent and seasonal oxygen‐deficient waters of the eastern South Pacific

The community structure of putative aerobic ammonia‐oxidizing archaea (AOA) was explored in two oxygen‐deficient ecosystems of the eastern South Pacific: the oxygen minimum zone off Peru and northern Chile (11°S–20°S), where permanent suboxic and low‐ammonium conditions are found at intermediate depths, and the continental shelf off central Chile (36°S), where seasonal oxygen‐deficient and relatively high‐ammonium conditions develop in the water column, particularly during the upwelling season. The AOA community composition based on the ammonia monooxygenase subunit A (amoA) genes changed according to the oxygen concentration in the water column and the ecosystem studied, showing a higher diversity in the seasonal low‐oxygen waters. The majority of the archaeal amoA genotypes was affiliated to the uncultured clusters A (64%) and B (35%), with Cluster A AOA being mainly associated with higher oxygen and ammonium concentrations and Cluster B AOA with permanent oxygen‐ and ammonium‐poor waters. Q‐PCR assays revealed that AOA are an abundant community (up to 10⁵amoA copies ml^?1), while bacterial amoA genes from β proteobacteria were undetected. Our results thus suggest that a diverse uncultured AOA community, for which, therefore, we do not have any physiological information, to date, is an important component of the nitrifying community in oxygen‐deficient marine ecosystems, and particularly in rich coastal upwelling ones.     

M. tuberculosis Pantothenate Kinase: Dual Substrate Specificity and Unusual Changes in Ligand Locations

Kinetic measurements of enzyme activity indicate that type I pantothenate kinase from Mycobacterium tuberculosis has dual substrate specificity for ATP and GTP, unlike the enzyme from Escherichia coli, which shows a higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes is provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate, (2) GDP and phosphopantothenate, (3) GDP, (4) GDP and pantothenate, (5) AMPPCP, and (6) GMPPCP, reported here, and the structures of the complexes of the two enzymes involving coenzyme A and different adenyl nucleotides reported earlier. The explanation is substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. The structures also provide a rationale for the movement of ligands during the action of the mycobacterial enzyme. Dual specificity of the type exhibited by this enzyme is rare. The change in locations of ligands during action, observed in the case of the M. tuberculosis enzyme, is unusual, so is the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands, and specificity. Furthermore, the dual specificity of the mycobacterial enzyme appears to have been caused by a biological necessity.     

Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B5). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP+ and pantoate bound, solved to 2.3 A of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn98 as well as Lys176 and Glu256 in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser244 for substrate binding and of Arg31 and Lys72 for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity.     

Structural insights into the stabilization of active,tetrameric DszC by its C‐terminus

Dibenzothiophene (DBT) is a typical sulfur‐containing compound found in fossil fuels. This compound and its derivatives are resistant to the hydrodesulfurization method often used in industry, but they are susceptible to enzymatic desulfurization via the 4S pathway, which is a well‐studied biochemical pathway consisting of four enzymes. DBT monooxygenase (DszC) from Rhodococcus erythropolis is involved in the first step of the 4S pathway. We determined the crystal structure of DszC, which reveals that, in contrast to several homologous proteins, the C‐terminus (410–417) of DszC participates in the stabilization of the substrate‐binding pocket. Analytical ultracentrifugation analysis and enzymatic assays confirmed that the C‐terminus is important for the stabilization of the active conformation of the substrate‐binding pocket and the tetrameric state. Therefore, the C‐terminus of DszC plays a significant role in the catalytic activity of this enzyme. Proteins 2014; 82:2733–2743. © 2014 Wiley Periodicals, Inc.     

Different growth trends of whitefish (Coregonus lavaretus) forms in the northern Baltic Sea

The sea growth of two whitefish forms, anadromous (Coregonus lavaretus lavaretus) and sea‐spawning (Coregonus lavaretus widegreni), was analysed using samples collected from the commercial sea catch in the Gulf of Bothnia (GoB) in the northern Baltic Sea during 1998–2014. In the GoB area, these two forms are possible to identify because the gill‐raker number and size at maturity vary between forms. The growth rate of the forms is linked to their feeding area. Sea‐spawning whitefish, which has a feeding migration near its home site, was shorter in the northern GoB (66°N–64°N) at the ages of 3–11 than those in the southern GoB (64°N–60°30′N). In the data, most whitefish were caught with gill nets in the GoB. The mesh sizes of gill nets capturing the anadromous form were mostly 35–45 mm, while those capturing the sea‐spawning form were <35 mm in the northern GoB. It is likely that the different growth trends for small and large whitefish were connected with differences in their recruitment for fishing. The length of anadromous females at the age of four sea years increased significantly, but the length of six‐year‐old anadromous female whitefish decreased over the catch years from 1998–2014. In contrast, the length of slow‐growing sea‐spawning whitefish of six years or older increased significantly in relation to the catch year in the gill‐net catch. The increase in the growth of young age groups in both forms was probably associated with the increasing temperature and the low fishing pressure on small fish. The decreasing age at capture for both forms and the depression of the mean size of old anadromous whitefish are signs of high fishing pressure with a high gill‐net effort that selectively removes the largest and oldest individuals of both forms.     

Structural and functional analyses of human tryptophan 2,3‐dioxygenase

Tryptophan 2,3‐dioxygenase (TDO), one of the two key enzymes in the kynurenine pathway, catalyzes the indole ring cleavage at the C2‐C3 bond of l ‐tryptophan. This is a rate‐limiting step in the regulation of tryptophan concentration in vivo, and is thus important in drug discovery for cancer and immune diseases. Here, we report the crystal structure of human TDO (hTDO) without the heme cofactor to 2.90 Å resolution. The overall fold and the tertiary assembly of hTDO into a tetramer, as well as the active site architecture, are well conserved and similar to the structures of known orthologues. Kinetic and mutational studies confirmed that eight residues play critical roles in l ‐tryptophan oxidation. Proteins 2014; 82:3210–3216. © 2014 Wiley Periodicals, Inc.     

The 2.1 Å crystal structure of an acyl‐CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl‐binding pocket for small branched acyl substrates

The acyl‐AMP forming family of adenylating enzymes catalyze two‐step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X‐ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl‐CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl‐CoA synthetase and 4‐chlorobenzoate:CoA ligase. Most interestingly, the acyl‐binding pocket of the new structure is compared with other acyl‐ and aryl‐CoA synthetases. A comparison of the acyl‐binding pocket of the acyl‐CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl‐binding pocket. Proteins 2009. © 2009 Wiley‐Liss, Inc.