Identification and functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681

Fusaricidin, a peptide antibiotic consisting of six amino acids, has been identified as a potential antifungal agent from Paenibacillus polymyxa. Here, we report the complete sequence of the fusaricidin synthetase gene (fusA) identified from the genome sequence of a rhizobacterium, P. polymyxa E681. The gene encodes a polypeptide consisting of six modules in a single open-reading frame. Interestingly, module six of FusA does not contain an epimerization domain, which suggests that the sixth amino acids of the fusaricidin analogs produced by P. polymyxa E681 may exist as an l-form, although all reported fusaricidins contain d-form alanines in their sixth amino acid residues. Alternatively, the sixth adenylation domain of the FusA may directly recognize the d-form alanine. The inactivation of fusA led to the complete loss of antifungal activity against Fusarium oxysporum. LC/MS analysis confirmed the incapability of fusaricidin production in the fusA mutant strain, thus demonstrating that fusA is involved in fusaricidin biosynthesis. Our findings suggested that FusA can produce more than one kind of fusaricidin, as various forms of fusaricidins were identified from P. polymyxa E681.     

Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics

Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket. Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of L-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide [Asn(5)]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.     

Cloning and characterization of a new hetero-gene cluster of nonribosomal peptide synthetase and polyketide synthase from the cyanobacterium Microcystis aeruginosa K-139

Two nonribosomal peptide synthetase genes responsible for the biosynthesis of microcystin and micropeptin in Microcystis aeruginosa K-139 have been identified. A new nonribosomal peptide synthetase gene, psm3, was identified in M. aeruginosa K-139. The gene is a cluster extending 30 kb and comprising 13 bidirectionally transcribed open reading frames arranged in two putative operons. psm3 encodes four adenylation proteins, one polyketide synthase, and several unique proteins, especially Psm3L consisting of halogenase, acyl-CoA binding protein-like protein, and acyl carrier protein. Alignment of the binding pocket of the adenylation domain and an ATP-PPi exchange analysis using a recombinant protein with the adenylation domain of Psm3B showed that Psm3G and Psm3B activate aspartic acid and tyrosine, respectively. Although disruption of psm3 did not reveal the product produced by Psm3, we identified microviridin B and aeruginosin K139 in the cells of M. aeruginosa K-139. The above-mentioned results indicated that M. aeruginosa possesses at least five nonribosomal peptide synthetase gene clusters.     

Tryptophan scanning mutagenesis in the TM3 domain of the Torpedo californica acetylcholine receptor beta subunit reveals an alpha-helical structure

We used tryptophan substitutions to characterize the beta M3 transmembrane domain (betaTM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the betaTM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [125I]-alpha-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the betaTM3 domain was consistent with an alpha-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed alpha-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica beta subunit.     

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that TM VIII faces an aqueous channel

The contribution of transmembrane region VIII of the Rickettsia prowazekii ATP/ADP translocase to the structure of the water-filled channel through which ATP is transported was evaluated from the accessibility of three hydrophilic, thiol reactive, methanethiosulfonate reagents to a library of 21 single-cysteine substitution mutants expressed in Escherichia coli. A negatively charged reagent (MTSES) and two positively charged reagents (MTSET and MTSEA) were used. Mutants Q323C and G327C did not tolerate cysteine substitution and were almost completely deficient in ATP transport. The remaining mutants exhibited 25-226% of the cysteine-less parent's transport activity. Five patterns of inhibition of ATP transport by the MTS reagents were observed. (i) ATP transport was not inhibited by any of the three MTS reagents in mutants Q321C, F324C, A332C, and L335C and only marginally in F333C. (ii) Transport activity of mutants F322C, Q326C, and A330C was markedly inhibited by all three reagents. (iii) ATP transport was inhibited by MTSEA in only the largest group of mutants (M334C, I336C, G337C, S338C, N339C, I340C, and I341C). (iv) Transport activity was inhibited by MTSET and MTSEA, whereas high concentrations of MTSES were required to inhibit mutants W328C, V329C, and I331C. However, mutant W328C could be inhibited by MTSES in the presence of sub-K(m) concentrations of the substrate. (v) ATP transport by mutant Y325C was unaffected by MTSEA, but inhibited approximately 50% by MTSET and MTSES. Transport of ATP protected mutants (F322C, W328C, V329C, A330C, and I331C) from MTS inhibition. Mutants in the half of TM VIII that is closest to the cytoplasm were not inhibited well by MTSES or MTSET in either whole cells or inside-out vesicles. The results indicate that TM VIII makes a major contribution to the structure of the aqueous translocation pathway, that the accessibility to impermeant thiol reagents is influenced (blocked or stimulated) by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.     

Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

Previous amino acid substitutions at the M4 domain of the Torpedo californica and mouse acetylcholine receptor suggested that the location of the substitution relative to the membrane-lipid interface and perhaps to the ion pore can be critical to the channel gating mechanism [Lasalde, J. A., Tamamizu, S., Butler, D. H., Vibat, C. R. T., Hung, B., and McNamee, M. G. (1996) Biochemistry 35, 14139-14148; Ortiz-Miranda, S. I., Lasalde, J. A., Pappone, P. A., and McNamee, M. G. (1997) J. Membr. Biol. 158, 17-30; Tamamizu, S., Lee, Y. H., Hung, B., McNamee, M. G., and Lasalde-Dominicci, J. A. (1999) J. Membr. Biol. 170, 157-164]. In this study, we introduce tryptophan substitutions at 12 positions (C412W, M415W, L416W, I417W, C418W, I419W, I420W, G421W, T422W, V423W, S424W, and V425W) along this postulated lipid-exposed segment M4 so that we can examine functional consequences on channel gating. The expression levels of mutants C412W, G421W, S424W, and V425W were almost the same as that of the wild type, whereas other mutants (M415W, L416W, C418W, I419W, I420W, T422W, and V423W) had relatively lower expression levels compared to that of the wild type as measured by iodinated alpha-bungarotoxin binding ([(125)I]-alpha-BgTx). Two positions (L416W and I419W) had less than 20% of the wild type expression level. I417W gave no detectable [(125)I]BgTx binding on the surface of oocyte, suggesting that this position might be involved in the AChR assembly, oligomerization, or transport to the cell membrane. The alphaV425W mutant exhibited a significant increase in the open channel probability with a moderate increase in the macroscopic response at higher ACh concentrations very likely due to channel block. The periodicity for the alteration of receptor assembly and ion channel function seems to favor a potential alpha-helical structure. Mutants that have lower levels of expression are clustered on one side of the postulated alpha-helical structure. Mutations that display normal expression and functional activity have been shown previously to face the membrane lipids by independent labeling studies. The functional analysis of these mutations will be presented and discussed in terms of possible structural models.     

Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor

Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC ₅₀, is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC ₅₀s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs. Received: 29 January 2001/Revised: 14 May 2001     

Book reviews

Book reviewed in this article:
HACCP: A P ractical A pproach (1994). By S. Mortimore and C. Wallace.
M olecular B iology of A rchaea (1994). Edited by F. Pfeiffer, P. Palm and K.-H. Schleifer.
R otaviruses (1994). Edited by R.F. Ramig.
P ower U nseen : H ow M icrobes R ule the W orld (1994). By B. Dixon.
V iruses and C ancer (1994). Edited by A. Minson, J. Niel and M. McCrae.
W ater M icrobiology (1994). By G. Bitton.
H andbook for R hizobia : M ethods in L egume - Rhizobium T echnology (1994). By P. Somasegaran and H.J. Hoben.
S tatistics and E xperimental D esign : A n I ntroduction for B iologists and B iochemists (1994). 3rd Edition. By G.M. Clarke
P rinciples of G ene M anipulation : A n I ntroduction to G enetic E ngineering (1994). 5th Edition. By R.W. Old and S.B. Primrose.
R espiratory I nfections : D iagnosis and M anagement (1994). Third edition. Edited by J.E. Pennington.
O bstetric and G ynecologic I nfectious D isease (1994). Edited by J.G. Pastorek II.
M olecular G enetics of B acteria (1994). 2nd Edition. By J.W. Dale.
B acterial P athogenesis : P art A: I dentification and R egulation of V irulence F actors (1994). Methods in Enzymology, Volume 235. Edited by V.L. Clark and P.M. Bavoil
B acterial P athogenesis : P art B: I nteraction of P athogenic B acteria with H ost C ells (1994). Methods in Enzymology, Volume 236. Edited by V.L. Clark and P.M. Bavoil.
M ononuclear P hagocytes : B iology of M onocytes and M acrophages (1992). Edited by R. van Furth.     

Book reviews

Book reviewed in this article:
B asic B iotechnology . A S tudent's G uide (1987). Edited by P. Prave, U. Faust, W. Sittig & D.A. Sukatsch.
B acteria AS P lant P athogens (1987). By Eve Billing.
B iotechnology. A C omprehensive T reatise I n 8 V olumes .
C urrent T opics I n M icrobiology A nd I mmunology 136: T he M olecular B iology O f B acterial V irus S ystems (1988). Edited by G. Hobom & R. Rott.
G enetic B iochemistry : F rom G ene T o P rotein (1988). By J. Etienne-Decant.
I ntracellular B acteria : C urrent T opics I n M icrobiology A nd I mmunology N o.
P roceedings O f T he 8TH I nternational B iotechnology S ymposium P aris 1988 (1989). Edited by G. Durand, L. Bobichon & J. Florent. Vol.
B iological W aste T reatment (1989). Edited by A. Mizrahi.
I n V itro T echniques I n R esearch (1989). Edited by J.W. Payne.
B loodstream I nfections : L aboratory D etection A nd C linical C onsiderations (1989). By C.L. Strand & J.A. Shulman.
M etal -M icrobe I nteraction (1989). Edited by Robert K. Poole & Geoffrey M. Gadd.
B iochemistry O f A ntimicrobial A ction (1989). By T.J. Franklin & G.A. Snow.
T heory A nd A pplication O f M icrobial A ssay (1989). By W. Hewitt & S. Vincent.     

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab)

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions.     

Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.     

Effects of deletion and site-directed mutations on ligation steps of NAD+-dependent DNA ligase: a biochemical analysis of BRCA1 C-terminal domain

DNA strand joining entails three consecutive steps: enzyme adenylation to form AMP-ligase, substrate adenylation to form AMP-DNA, and nick closure. In this study, we investigate the effects on ligation steps by deletion and site-directed mutagenesis of the BRCA1 C-terminal (BRCT) domain using NAD(+)-dependent DNA ligase from Thermus species AK16D. Deletion of the BRCT domain resulted in substantial loss of ligation activity, but the mutant was still able to form an AMP-ligase intermediate, suggesting that the defects caused by deletion of the entire BRCT domain occur primarily at steps after enzyme adenylation. The lack of AMP-DNA accumulation by the domain deletion mutant as compared to the wild-type ligase indicates that the BRCT domain plays a role in the substrate adenylation step. Gel mobility shift analysis suggests that the BRCT domain and helix-hairpin-helix subdomain play a role in DNA binding. Similar to the BRCT domain deletion mutant, the G617I mutant showed a low ligation activity and lack of accumulation of AMP-DNA intermediate. However, the G617I mutant was only weakly adenylated, suggesting that a point mutation in the BRCT domain could also affect the enzyme adenylation step. The significant reduction of ligation activity by G634I appears to be attributable to a defect at the substrate adenylation step. The greater ligation of mismatched substrates by G638I is accountable by accelerated conversion of the AMP-DNA intermediate to a ligation product at the final nick closure step. The mutational effects of the BRCT domain on ligation steps in relation to protein-DNA and potential protein-protein interactions are discussed.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

REVIEWS

Review in this article:
Outline of Palaeobotany. By W ładysław S zafer and M ikołaj K ostyniuk .
[ Zarys Palaeobotaniki. W ładysław S zafer i M ikołaj K ostyniuk .]
Annual Review of Plant Physiology , Vol. V. Edited by D. J. A rnon and L. M achlis .
Analytic Studies in Plant Respiration. By the late F. F. B lackman .
Cold Spring Harbor Symposia on Quantitative Biology. Vol. xviii. Viruses.
Plant Life in Malaya. By R. E. H olttum .
The Changing Flora of Britain. By J. E. L ousley .     

Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp–hydroxamate–Fe³⁺ complex and the formation of l-Trp–l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.     

Tryptophan scanning mutagenesis in the alphaM3 transmembrane domain of the Torpedo californica acetylcholine receptor: functional and structural implications

The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC(50). Taken together, our results suggest that alphaM3 contributes to the gating machinery of the nicotinic ACh receptor and that alphaM3 is comprised of a mixture of two types of helical structures.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Inactivation of the phosphoglucomutase gene pgm in Paenibacillus polymyxa leads to overproduction of fusaricidin

Fusaricidin, a lipodepsipeptide isolated from Paenibacillus polymyxa, has high antimicrobial activity against fungi and Gram-positive bacteria. Through mutagenesis, we obtained two mutant strains, N1U7 and N17U7, which produce 6.2- to 7.9-fold more fusaricidin than their parent strain. Causal mutations were identified by whole-genome sequencing, and the two strains each contained at least eleven point mutations, including four common mutations. A mutation in the PPE04441 gene (pgm), encoding an α-phosphoglucomutase, was found to be an important factor in fusaricidin overproduction by complementation experiments. Null mutation of pgm in the parental strain increased fusaricidin production by 5.2-fold. Increased growth and cell viability in stationary phase, reduced exopolysaccharide production, and increased fusA expression were observed in the pgm mutant strains, which might be related to fusaricidin overproduction. This is the first report revealing that PGM deficiency leads to an overproduction of fusaricidin.     

耳聋相关基因COCH的非同义单核苷酸多态性致聋表型预测

群体凝血因子C同源物基因(Coagulation factor C homology,COCH)是人类发现的第一个伴前庭功能障碍的耳聋基因,位于人类染色体14q12-q13上。迄今,在COCH基因上发现16个位点突变导致常染色体显性遗传非综合征型耳聋DFNA9的发生,其中包括13个非同义单核苷酸多态性(Non-synonymous single nucleotide polymorphisms,nsSNPs)位点。由于该基因其他nsSNPs的基因型与表型关系尚不清楚,因此文章采用生物信息学方法,从COCH基因全部的SNPs中分级筛选,结合已知的致病nsSNPs信息及蛋白三维结构验证,首次预测出由COCH基因编码的cochlin蛋白的vWFA (Von Willebrand factor type A domain)区的8个高风险致病性nsSNPs(I176T、R180Q、G265E、V269L、I368N、I372T、R416C和Y424D)。同时,对位于LCCL (Limulus factor C, cochlin, and late gestation lung protein Lgl1)区域的6个已知致病突变的nsSNPs ( P51S、G87W、I109N、I109T、W117R和F121S)进行了三维结构模拟,发现突变体均发生了环状结构或链状结构的改变。本研究对COCH基因的基因型与表型的相关性研究为遗传性耳聋筛查提供了相应的理论依据,也对该基因所编码的cochlin蛋白的功能研究具有一定的指导意义。