Specificity profile of NAT/NCS2 purine transporters in Sinorhizobium (Ensifer) meliloti

Sinorhizobium (Ensifer) meliloti is a model example of a soil alpha-proteobacterium which induces the formation of nitrogen-fixing symbiotic nodules on the legume roots. In contrast to all other rhizobacterial species, S. meliloti contains multiple homologs of nucleobase transporter genes that belong to NAT/NCS2 family (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2). We analyzed functionally all (six) relevant homologs of S. meliloti 1,021 using Escherichia coli K-12 as a host and found that five of them are high-affinity transporters for xanthine (SmLL9), uric acid (SmLL8, SmLL9, SmX28), adenine (SmVC3, SmYE1), guanine (SmVC3), or hypoxanthine (SmVC3). Detailed analysis of substrate profiles showed that two of these transporters display enlarged specificity (SmLL9, SmVC3). SmLL9 is closely related in sequence with the xanthine-specific XanQ of E. coli. We subjected SmLL9 to rationally designed site-directed mutagenesis and found that the role of key binding-site residues of XanQ is conserved in SmLL9, whereas a single amino-acid change (S93N) converts the xanthine/uric-acid transporter SmLL9 to a xanthine-preferring variant, due to disruption of an essential hydrogen bond with the C8 oxygen of uric acid. The results highlight the presence of several different purine nucleobase transporters in S. meliloti and imply that the purine transport might be important in the nodule symbiosis involving S. meliloti.     

Functional Identification of the Hypoxanthine/Guanine Transporters YjcD and YgfQ and the Adenine Transporters PurP and YicO of Escherichia coli K-12

The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N⁶-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.     

The role of transmembrane segment TM3 in the xanthine permease XanQ of Escherichia coli

The xanthine permease XanQ of Escherichia coli is used as a study prototype for function-structure analysis of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family. Our previous mutagenesis study of polar residues of XanQ has shown that Asn-93 at the middle of putative TM3 is a determinant of substrate affinity and specificity. To study the role of TM3 in detail we employed Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 79-107 (YGIVGSGLLSIQSVNFSFVTVMIALGSSM) including TM3 (underlined) and flanking sequences was replaced individually with Cys. Of 29 single-Cys mutants, 20 accumulate xanthine to 40-110% of the steady state observed with C-less, six (S88C, F94C, A102C, G104C, S106C) accumulate to low levels (10-30%) and three (G83C, G85C, N93C) are inactive. Extensive mutagenesis reveals that Gly-83 and, to a lesser extent, Gly-85, are crucial for expression in the membrane. Replacements of Asn-93 disrupt affinity (Thr) or permit recognition of 8-methylxanthine which is not a wild-type ligand (Ala, Ser, Asp) and utilization of uric acid which is not a wild-type substrate (Ala, Ser). Replacements of Phe-94 impair affinity for 2-thio and 6-thioxanthine (Tyr) or 3-methylxanthine (Ile). Single-Cys mutants S84C, L86C, L87C, and S95C are highly sensitive to inactivation by N-ethylmaleimide. Our data reveal that key residues of TM3 cluster in two conserved sequence motifs, (83)GSGLL(87) and (93)NFS(95), and highlight the importance of Asn-93 and Phe-94 in substrate recognition and specificity; these findings are supported by structural modeling on the recently described x-ray structure of the uracil-transporting homolog UraA.     

Insights to the evolution of Nucleobase-Ascorbate Transporters (NAT/NCS2 family) from the Cys-scanning analysis of xanthine permease XanQ

The nucleobase-ascorbate transporter or nucleobase-cation symporter-2 (NAT/NCS2) family is one of the five known families of transporters that use nucleobases as their principal substrates and the only one that is evolutionarily conserved and widespread in all major taxa of organisms. The family is a typical paradigm of a group of related transporters for which conservation in sequence and overall structure correlates with high functional variations between homologs. Strikingly, the human homologs fail to recognize nucleobases or related cytotoxic compounds. This fact allows important biomedical perspectives for translation of structure-function knowledge on this family to the rational design of targeted antimicrobial purine-related drugs. To date, very few homologs have been characterized experimentally in detail and only two, the xanthine permease XanQ and the uric acid/xanthine permease UapA, have been studied extensively with site-directed mutagenesis. Recently, the high-resolution structure of a related homolog, the uracil permease UraA, has been solved for the first time with crystallography. In this review, I summarize current knowledge and emphasize how the systematic Cys-scanning mutagenesis of XanQ, in conjunction with existing biochemical and genetic evidence for UapA and the x-ray structure of UraA, allow insight on the structure-function and evolutionary relationships of this important group of transporters. The review is organized in three parts referring to (I) the theory of use of Cys-scanning approaches in the study of membrane transporter families, (II) the state of the art with experimental knowledge and current research on the NAT/NCS2 family, (III) the perspectives derived from the Cys-scanning analysis of XanQ.     

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family

NCS1 proteins are H⁺ or Na⁺ symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur‐ and Fcy‐like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5‐fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate‐affinity, low‐capacity, highly specific adenine transporter, whereas FcyE contributes to 8‐azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.     

Melain G, a cysteine protease from green fruits of the bead tree, Melia azedarach: a protease affected by specific amino acids at P3 position

A protease (melain G) was isolated from the greenish fruits of the bead tree, Melia azedarach var. japonica Makino. Melain G shares 110 identical amino acid residues (50%) with papain, 112 (51%) with actinidain, and 91 (41%) with stem bromelain. From the sites cleaved in the oxidized insulin B-chain and synthetic oligopeptide substrates by melain G, the enzyme preferred small amino acid residues such as Gly or Ser at the P2 position and negatively charged residues such as glutamic or cysteic acid at the P3 position. This is clearly different from the specificity of papain, which prefers the large hydrophobic amino acid residues such as Phe, Val, and Leu at the P2 position. Accordingly, it is presumed that the bottom of the S2 pocket of melain G is shallow due to the presence of a Phe residue, and a bulky P2 substrate (for example Phe residue) is not preferred by the enzyme. Negatively charged residues at the P3 position of substrates well suited the S3 site of melain G for making a salt bridge. It is likely that Arg61 is the S3 position of melain G by analogy with papain.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Roles of Tyr13 and Phe219 in the unique substrate specificity of pepsin B

Pepsin B is known to be distributed throughout mammalia, including carnivores. In this study, the proteolytic specificity of canine pepsin B was clarified with 2 protein substrates and 37 synthetic octapeptides and compared with that of human pepsin A. Pepsin B efficiently hydrolyzed gelatin but very poorly hydrolized hemoglobin. It was active against only a group of octapeptides with Gly at P2, such as KPAGF/LRL and KPEGF/LRL (arrows indicate cleavage sites). In contrast, pepsin A hydrolyzed hemoglobin but not gelatin and showed high activity against various types of octapeptides, such as KPAEF/FRL and KPAEF/LRL. The specificity of pepsin B is unique among pepsins, and thus, the enzyme provides a suitable model for analyzing the structure and function of pepsins and related aspartic proteinases. Because Tyr13 and Phe219 in/around the S2 subsites (Glu/Ala13 and Ser219 are common in most pepsins) appeared to be involved in the specificity of pepsin B, site-directed mutagenesis was undertaken to replace large aromatic residues with small residues and vice versa. The Tyr13Ala/Phe219Ser double mutant of pepsin B was found to demonstrate broad activity against hemoglobin and various octapeptides, whereas the reverse mutant of pepsin A had significantly decreased activity. According to molecular modeling of pepsin B, Tyr13 OH narrows the substrate-binding space and a peptide with Gly at P2 might be preferentially accommodated because of its high flexibility. The hydroxyl can also make a hydrogen bond with nitrogen of a P3 residue and fix the substrate main chain to the active site, thus restricting the flexibility of the main chain and strengthening preferential accommodation of Gly at P2. The phenyl moiety of Phe219 is bulky and narrows the S2 substrate space, which also leads to a preference for Gly at P2, while lowering the catalytic activity against other peptide types without making a hydrogen-bonding network in the active site.     

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-6³-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

An essential residue in the flexible peptide linking the two idiosynchratic domains of bacterial tyrosyl-tRNA synthetases

Tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus comprises three sequential domains: an N-terminal catalytic domain, an alpha-helical domain with unknown function, and a C-terminal tRNA binding domain (residues 320-419). The properties of the polypeptide segment that links the alpha-helical and C-terminal domains, were analyzed by measuring the effects of sequence changes on the aminoacylation of tRNA(Tyr) with tyrosine. Mutations F323A (Phe323 into Ala), S324A, and G325A showed that the side chain of Phe323 was essential but not those of Ser324 and Gly325. Insertions of Gly residues between Leu322 and Phe323 and the point mutation L322P showed that the position and precise orientation of Phe323 relative to the alpha-helical domain were important. Insertions of Gly residues between Gly325 and Asp326 and deletion of residues 330-339 showed that the length and flexibility of the sequence downstream from Gly325 were unimportant but that this sequence could not be deleted. Mutations F323A, -L, -Y, and -W showed that the essential property of Phe323 was its aromaticity. The Phe323 side chain contributed to the stability of the initial complex between TyrRS and tRNA(Tyr) for 2.0 +/- 0.2 kcal x mol(-1) and to the stability of their transition state complex for 4.2 +/- 0.1 kcal x mol(-1), even though it is located far from the catalytic site. The results indicate that the disorder of the C-terminal domain in the crystals of TyrRS is due to the flexibility of the peptide that links it to the helical domain. They identified Phe323 as an essential residue for the recognition of tRNA(Tyr).     

Construction and expression of mutant cDNAs responsible for genetic polymorphism in aldehyde oxidase in Donryu strain rats

We demonstrated the genetic polymorphism of aldehyde oxidase (AO) in Donryu strain rats: the ultrarapid metabolizer (UM) with nucleotide mutation of (377G, 2604C) coding for amino acid substitution of (110Gly, 852Val), extensive metabolizer (EM) with (377G/A, 2604C/T) coding for (110Gly/Ser, 852Val/Ala), and poor metabolizer (PM) with (377A, 2604T) coding for (110Ser, 852Ala), respectively. The results suggested that 377G > A and/or 2604C > T should be responsible for the genetic polymorphism. In this study, we constructed an E. coli expression system of four types of AO cDNA including Mut-1 with (377G, 2604T) and Mut-2 with (377A, 2604C) as well as naturally existing nucleotide sequences of UM and PM in order to clarify which one is responsible for the polymorphism. Mut-1 and Mut-2 showed almost the same high and low activity as that of the UM and PM groups, respectively. Thus, the expression study of mutant AO cDNA directly revealed that the nucleotide substitution of 377G > A, but not that of 2604C > T, will play a critical role in the genetic polymorphism of AO in Donryu strain rats. The reason amino acid substitution will cause genetic polymorphism in AO activity was discussed.     

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F₁ with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F₁ sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β_DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β_E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.     

Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase

Recently, two l-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward l-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K _m values. Xt-F143Y caused a higher increase in the K _m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to l-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.     

Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

Identification of Escherichia coli Mismatch-specific Uracil DNA Glycosylase as a Robust Xanthine DNA Glycosylase

The gene for the mismatch-specific uracil DNA glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the human thymine DNA glycosylase with activity against mismatched uracil base pairs. Examination of cell extracts led us to detect a previously unknown xanthine DNA glycosylase (XDG) activity in E. coli. DNA glycosylase assays with purified enzymes indicated the novel XDG activity is attributable to MUG. Here, we report a biochemical characterization of xanthine DNA glycosylase activity in MUG. The wild type MUG possesses more robust activity against xanthine than uracil and is active against all xanthine-containing DNA (C/X, T/X, G/X, A/X and single-stranded X). Analysis of potentials of mean force indicates that the double-stranded xanthine base pairs have a relatively narrow energetic difference in base flipping, whereas the tendency for uracil base flipping follows the order of C/U > G/U > T/U > A/U. Site-directed mutagenesis performed on conserved motifs revealed that Asn-140 and Ser-23 are important determinants for XDG activity in E. coli MUG. Molecular modeling and molecular dynamics simulations reveal distinct hydrogen-bonding patterns in the active site of E. coli MUG that account for the specificity differences between E. coli MUG and human thymine DNA glycosylase as well as that between the wild type MUG and the Asn-140 and Ser-23 mutants. This study underscores the role of the favorable binding interactions in modulating the specificity of DNA glycosylases.     

Substrate selectivity of YgfU, a uric acid transporter from Escherichia coli

The ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family includes more than 2,000 members, but only 15 have been characterized experimentally. Escherichia coli has 10 members, of which the uracil permease UraA and the xanthine permeases XanQ and XanP are functionally known. Of the remaining members, YgfU is closely related in sequence and genomic locus with XanQ. We analyzed YgfU and showed that it is a proton-gradient dependent, low-affinity (K(m) 0.5 mM), and high-capacity transporter for uric acid. It also shows a low capacity for transport of xanthine at 37 °C but not at 25 °C. Based on the set of positions delineated as important from our previous Cys-scanning analysis of permease XanQ, we subjected YgfU to rationally designed site-directed mutagenesis. The results show that the conserved His-37 (TM1), Glu-270 (TM8), Asp-298 (TM9), and Gln-318 and Asn-319 (TM10) are functionally irreplaceable, and Thr-100 (TM3) is essential for the uric acid selectivity because its replacement with Ala allows efficient uptake of xanthine. The key role of these residues is corroborated by the conservation pattern and homology modeling on the recently described x-ray structure of permease UraA. In addition, site-specific replacements at TM8 (S271A, M274D, V282S) impair expression in the membrane, and V320N (TM10) inactivates the permease, whereas R327G (TM10) or S426N (TM14) reduces the affinity for uric acid (4-fold increased K(m)). Our study shows that comprehensive analysis of structure-function relationships in a newly characterized transporter can be accomplished with relatively few site-directed replacements, based on the knowledge available from Cys-scanning mutagenesis of a prototypic homolog.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.