BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

Structural Changes in the Catalytic Cycle of the Na,K-ATPase Studied by Infrared Spectroscopy

Pig kidney Na⁺,K⁺-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na₃⁺ to an E2P state was initiated by photolysis of P³-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg²⁺ and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na⁺,K⁺-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15°C. Several controls ensured that the observed difference signals were due to structural changes of the Na⁺,K⁺-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca²⁺-ATPase and H⁺,K⁺-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm⁻¹, respectively, for αβ heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na₃⁺ to an E2P state. A negative band at 1730 cm⁻¹ is in line with the presence of a protonated Asp or Glu residue that coordinates Na⁺ in E1Na₃⁺. Infrared signals were also detected in the absorption regions of ionized carboxyl groups.     

Functional significance of some particular amino acid residues in Bombyx mori pyridoxal kinase

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B₆ metabolism in animals. It catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal 5′-phosphate, an important cofactor for many enzymatic reactions. Bombyx mori PLK (BmPLK) is 10 or more residues shorter than mammalian PLKs, and some amino acid residues conserved in the PLKs from mammals are not maintained in the protein. Multiple sequence alignment suggested that amino acid residues Thr⁴⁷, Ile⁵⁴, Arg⁸⁸, Asn¹²¹ and Glu²³⁰ might play important roles in BmPLK. In this study, we used a site-directed specific mutagenesis approach to determine the functional significance of these particular amino acid residues in BmPLK. Our results demonstrated that the mutation of Asn¹²¹ to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr⁴⁷ to Asn, Ile⁵⁴ to Phe, and Arg⁸⁸ to Ile displayed a decreased catalytic efficiency and an elevated Km value for substrate relative to the wild-type value, and no enzyme activity could be detected in mutant of Trp²³⁰ to Glu. Circular dichroism analysis revealed that the mutation of Trp²³⁰ to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp²³⁰ is crucial to maintain the structural and functional integrity of BmPLK. This study will add to the existing understanding of the characteristic of structure and function of BmPLK.     

家蚕酪氨酸羟化酶基因BmTh的表达及功能

酪氨酸羟化酶作为儿茶酚胺合成的限速酶, 广泛存在于昆虫、哺乳动物和人类中, 是其新陈代谢不可缺少的酶类。在其他昆虫中, 酪氨酸羟化酶参与了黑色素的合成, 并在昆虫外骨骼的硬化过程中发挥关键作用。为了研究家蚕Bombyx mori酪氨酸羟化酶基因的生理生化功能, 本文对其基因结构、表达特征及功能进行了研究。基于家蚕基因组和基因芯片数据的生物信息学分析表明, BmTh位于家蚕1号染色体上, 含有8个外显子, 编码561个氨基酸。基因芯片数据显示在家蚕5龄第3天的头部和体壁组织中的表达量较高, RT-PCR验证结果与此一致。利用石蜡组织切片材料和RNA探针对BmTh进行表达定位, 原位杂交结果显示在家蚕头部边缘和体壁上有明显的杂交信号。在幼虫发育至熟蚕时注射酪氨酸羟化酶抑制剂3-indole-L-tyrosine （3-IT）, 20 mmol/L的浓度对幼虫几乎没有影响, 50 mmol/L的浓度导致幼虫变态不完全和化蛹困难, 100 mmol/L的浓度使幼虫致死且体色变黑。结果提示, BmTh对家蚕变态发育起重要作用, 是家蚕正常发育不可缺少的关键基因。     

Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

The iron atom in the nonheme iron monooxygenase phenylalanine hydroxylase is bound on one face by His285, His290, and Glu330. This arrangement of metal ligands is conserved in the other aromatic amino acid hydroxylases, tyrosine hydroxylase and tryptophan hydroxylase. A similar 2-His-1-carboxylate facial triad of two histidines and an acidic residue are the ligands to the iron in other nonheme iron enzymes, including the α-ketoglutarate-dependent hydroxylases and the extradiol dioxygenases. Previous studies of the effects of conservative mutations of the iron ligands in tyrosine hydroxylase established that there is some plasticity in the nature of the ligands and that the three ligands differ in their sensitivity to mutagenesis. To determine the generality of this finding for enzymes containing a 2-His-1-carboxylate facial triad, the His285, His290, and Glu330 in rat phenylalanine hydroxylase were mutated to glutamine, glutamate, and histidine. All of the mutant proteins had low but measurable activities for tyrosine formation. In general, mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All of the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. The H285Q enzyme had the highest activity as tetrahydropterin oxidase at 20% the wild-type value. All of the mutations greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family.     

Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π¹ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.     

Crystal Structure of the Catalytic Domain of the Bacillus cereus SleB Protein, Important in Cortex Peptidoglycan Degradation during Spore Germination

The SleB protein is one of two redundant cortex-lytic enzymes (CLEs) that initiate the degradation of cortex peptidoglycan (PG), a process essential for germination of spores of Bacillus species, including Bacillus anthracis. SleB has been characterized as a soluble lytic transglycosylase that specifically recognizes spore cortex PG and catalyzes the cleavage of glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine residues with concomitant formation of a 1,6-anhydro bond in the NAM residue. We found that like the full-length Bacillus cereus SleB, the catalytic C-terminal domain (SleB^C) exhibited high degradative activity on cortex PG in vitro, although SleB''s N-terminal domain, thought to bind PG, was inactive. The 1.85-Å crystal structure of SleB^C reveals an ellipsoid molecule with two distinct domains dominated by either α helices or β strands. The overall fold of SleB closely resembles that of the catalytic domain of the family 1 lytic transglycosylases but with a completely different topological arrangement. Structural analysis shows that an invariant Glu157 of SleB is in a position equivalent to that of the catalytic glutamate in other lytic transglycosylases. Indeed, SleB bearing a Glu157-to-Gln mutation lost its cortex degradative activity completely. In addition, the other redundant CLE (called CwlJ) in Bacillus species likely has a three-dimensional structure similar to that of SleB, including the invariant putative catalytic Glu residue. SleB and CwlJ may offer novel targets for the development of anti-spore agents.     

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide ¹H/²H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.     

Lambda CIN-1, a New Mutation Which Enhances Lysogenization by Bacteriophage Lambda, and the Genetic Structure of the Lambda CY Region

Seven lambda cy mutants have been mapped within a small region located approximately halfway between the rightward boundary of the imm⁴³⁴ region and the lambda cII gene. The seven mutants lie at four sites separated by a total distance of about 12 nucleotide pairs, as estimated from recombination frequencies. Six of the seven mutants lie on the right side of the cy fine structure map, spanning a total distance of about 3–5 nucleotide pairs. Lying approximately 11–21 nucleotide pairs to the left of the leftmost cy mutant is a newly described mutation called cin-1, for c independent. The cin-1 mutation allows some lysogenization when coupled with any cy, cII or cIII mutant, but not when coupled with a defective cI gene. The cin-1 mutation, like cy mutants, has a cis-dominant action upon the cI gene in mixed infections. The observation that λimm⁴³⁴ cin-1 cy2001 lysogenizes efficiently, but not λimm⁴³⁴ cin-1 cy2001 cII68 nor any other λimm⁴³⁴ cin-1 cy derivative, is interpreted to mean that all of the cy mutants on the right side of the cy fine structure map inactivate a binding site for cII/cIII function, but that cy2001, the single mutant on the left side of the cy fine structure map, does not inactivate that binding site.     

Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6₅22 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P2₁2₁2₁, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.     

Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2′-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37→Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T→C:G transversion mutation in a mutT^– mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.     

A Mutation in the N Domain of Escherichia coli Lon Stabilizes Dodecamers and Selectively Alters Degradation of Model Substrates

Escherichia coli Lon, an ATP-dependent AAA⁺ protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, Lon^E240K exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, Lon^E240K displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity.     

Transmembrane Domain Sequence Requirements for Activation of the p185c-neu Receptor Tyrosine Kinase

The receptor tyrosine kinase p185^c-neu can be constitutively activated by the transmembrane domain mutation Val⁶⁶⁴→ Glu, found in the oncogenic mutant p185^neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185^c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185^c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]_n or [VVVEVVV]_n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.     

Analysis of conserved glutamate residues in Porphyromonas gingivalis outer membrane receptor HmuR: toward a further understanding of heme uptake

The aim of this study was to broaden the current knowledge about the Porphyromonas gingivalis heme receptor HmuR. Site-directed mutagenesis was employed to replace Glu427, Glu448, Glu458 and Glu503 by alanines and to construct a triple Glu427Ala/Glu448Ala/Glu 458Ala mutant. All iron/heme-starved P. gingivalis mutants showed decreased growth recovery when human serum as the iron/heme source was used, hmuR::ermF, hmuR ^E503A and hmuR ^{E427A,E448A,E458A} mutant strains being the most affected. E. coli cells expressing HmuR with mutated glutamate residues bound hemin, hemoglobin and hemin–serum albumin complex with the same efficiency as did the wild-type recombinant protein, suggesting that the residues were not directly involved in heme binding. These data indicate that in addition to two conserved histidine residues (His95 and His434), NPDL and YRAP motifs, conserved glutamate residues are important for HmuR to utilize heme present in serum hemoproteins.     

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α₁-antitrypsin (α₁AT) deficiency cases. It leads to increased polymerization of the serpin α₁AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α₁AT and analyzed their fluorescence properties. α₁AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α₁AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α₁AT compared to M α₁AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B.     

Temperature-sensitive mutants of the EcoRI endonuclease

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30°C and 42°C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 Å resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.     

Crystal Structure and Functional Analysis of the Glutaminyl Cyclase from Xanthomonas campestris

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-Å resolution. The structure shows a five-bladed β-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu⁴⁵) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu⁸⁹ as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of β-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes.     

ENU mutagenesis identifies mice with morbid obesity and severe hyperinsulinemia caused by a novel mutation in leptin

Background

Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals.

Methodology/Principal Findings

To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin^145E/145E mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin^145E/145E mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin^145E/145E mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region.

Conclusions/Significance

Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome.