Structure of the N‐terminal domain of the metalloprotease PrtV from Vibrio cholerae

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N‐terminal domain (residues 23–103) that contains two short α‐helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C‐terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.     

Sequence fingerprint and structural analysis of the SCOR enzyme A3DFK9 from Clostridium thermocellum

We have identified a highly conserved fingerprint of 40 residues in the TGYK subfamily of the short‐chain oxidoreductase enzymes. The TGYK subfamily is defined by the presence of an N‐terminal TGxxxGxG motif and a catalytic YxxxK motif. This subfamily contains more than 12,000 members, with individual members displaying unique substrate specificities. The 40 fingerprint residues are critical to catalysis, cofactor binding, protein folding, and oligomerization but are substrate independent. Their conservation provides critical insight into evolution of the folding and function of TGYK enzymes. Substrate specificity is determined by distinct combinations of residues in three flexible loops that make up the substrate‐binding pocket. Here, we report the structure determinations of the TGYK enzyme A3DFK9 from Clostridium thermocellum in its apo form and with bound NAD⁺ cofactor. The function of this protein is unknown, but our analysis of the substrate‐binding loops putatively identifies A3DFK9 as a carbohydrate or polyalcohol metabolizing enzyme. C. thermocellum has potential commercial applications because of its ability to convert biomaterial into ethanol. A3DFK9 contains 31 of the 40 TGYK subfamily fingerprint residues. The most significant variations are the substitution of a cysteine (Cys84) for a highly conserved glycine within a characteristic VNNAG motif, and the substitution of a glycine (Gly106) for a highly conserved asparagine residue at a helical kink. Both of these variations occur at positions typically participating in the formation of a catalytically important proton transfer network. An alternate means of stabilizing this proton wire was observed in the A3DFK9 crystal structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis

M.EcoHK31I is a naturally occurring mC5-methyltransferase with a large alpha polypeptide and a small beta polypeptide. Polypeptide alpha contains conserved motifs I-VIII and X, and polypeptide beta contains motif IX. To understand how polypeptide alpha carries out its function, a molecular model of the large domain of polypeptide alpha was generated using M.HhaI and M.HaeIII as templates. The large domain is a mixed alpha/beta structure. Residues 15-19 in motif I (Phe-Naa-Gly-Naa) are conserved for cofactor binding. The key catalytic residue Cys-79 in motif IV is also conserved in comparison with other C-5 MTases. Comparing polypeptide alpha with M.HhaI and M.HaeIII revealed a unique region upstream of motif X. To understand the role of this region, 14 charged residues between R224 and E271 in the putative small domain were mutated. Activity assays indicated that most of these charges can be eliminated or changed conservatively. Among these charged residues, R224, E240, D245 and D251 may take part in proper interaction with DNA in the presence of polypeptide beta.     

Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA

In thermophilic bacteria, specific 2‐thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2‐thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur‐carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2‐thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N‐ and C‐terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA^Ile₂ lysidine synthetase), than to the other type of tRNA 2‐thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP‐binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA‐binding site. Proteins 2013; 81:1232–1244. © 2013 Wiley Periodicals, Inc.     

X‐ray crystal structure of the N‐terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR_1–105) and 8–105 (NTR_8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR_1–105 packs as a dimer and NTR_8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn²⁺ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn²⁺ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc.     

Processing and targeting of cathepsin L (TbCatL) to the lysosome in Trypanosoma brucei

Cathepsin L (TbCatL) is an essential lysosomal thiol protease in African trypanosomes. TbCatL is synthesized as two precursor forms (P/X) that are activated to mature form (M) with the removal of the prodomain upon arrival in the lysosome. We examine TbCatL trafficking in a novel system: truncated TbCatL reporter without the C‐terminal domain (CTD; TbCatL?) ectopically expressed in an RNA interference (RNAi) cell line targeting the CTD/3′ untranslated region (UTR) of endogenous mRNA. TbCatL? is synthesized as P′/X′/M′ species, localizes to the lysosome, and rescues the lethal TbCatL RNAi phenotype. Inactive TbCatLΔ:C150A is only processed to M′ in the presence of endogenous TbCatL indicating trans‐auto‐catalytic activation. X′ is formed with active endoplasmic reticulum (ER)‐retained TbCatLΔ:MDDL, but not with TbCatLΔ:C150A, indicating stochastic generation in the ER by cis‐auto‐cleavage within the prodomain of newly synthesized P′. Modelling the TbCatL prodomain on the human CatL structure suggests three solvent accessible features that could contain post‐Golgi targeting signals: the N‐terminus, the helix 1/turn 1 junction, and a separate turn (T3). We demonstrate that the critical motif for lysosomal targeting is an asparagine‐proline dipeptide in T3 that is strictly conserved in all Kinetoplastida. These findings show novel insights on the maturation of TbCatL, which is a critical virulence factor in mammalian infection.     

Insulin-regulated aminopeptidase: analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine beta-naphthylamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.     

Identification of the amino acid residues and domains in the cysteine‐rich protein of Chinese wheat mosaic virus that are important for RNA silencing suppression and subcellular localization

Cysteine‐rich proteins (CRPs) encoded by some plant viruses in diverse genera function as RNA silencing suppressors. Within the N‐terminal portion of CRPs encoded by furoviruses, there are six conserved cysteine residues and a Cys–Gly–X–X–His motif (Cys, cysteine; Gly, glycine; His, histidine; X, any amino acid residue) with unknown function. The central domains contain coiled‐coil heptad amino acid repeats that usually mediate protein dimerization. Here, we present evidence that the conserved cysteine residues and Cys–Gly–X–X–His motif in the CRP of Chinese wheat mosaic virus (CWMV) are critical for protein stability and silencing suppression activity. Mutation of a leucine residue in the third coiled‐coil heptad impaired CWMV CRP activity for suppression of local silencing, but not for the promotion of cell‐to‐cell movement of Potato virus X (PVX). In planta and in vitro analysis of wild‐type and mutant proteins indicated that the ability of the CRP to self‐interact was correlated with its suppression activity. Deletion of up to 40 amino acids at the C‐terminus did not abolish suppression activity, but disrupted the association of CRP with endoplasmic reticulum (ER), and reduced its activity in the enhancement of PVX symptom severity. Interestingly, a short region in the C‐terminal domain, predicted to form an amphipathic α‐helical structure, was responsible for the association of CWMV CRP with ER. Overall, our results demonstrate that the N‐terminal and central regions are the functional domains for suppression activity, whereas the C‐terminal region primarily functions to target CWMV CRP to the ER.     

Structural analysis of CPF_2247, a novel α-amylase from Clostridium perfringens

CPF_2247 from Clostridium perfringens ATCC 13124 was identified as a putative carbohydrate‐active enzyme by its low sequence identity to endo‐β‐1,4‐glucanases belonging to family 8 of the glycoside hydrolase classification. The X‐ray crystal structure of CPF_2247 determined to 2.0 Å resolution by single‐wavelength anomalous dispersion using seleno‐methionine‐substituted protein revealed an (α/α)₆ barrel fold. A large cleft on the surface of the protein contains residues that are structurally conserved with key elements of the catalytic machinery in clan GH‐M glycoside hydrolases. Assessment of CPF_2247 as a carbohydrate‐active enzyme disclosed α‐glucanase activity on amylose, glycogen, and malto‐oligosaccharides. Proteins 2011;. © 2011 Wiley‐Liss, Inc.     

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation‐PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C‐terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. Proteins 2014; 82:2691–2702. © 2014 Wiley Periodicals, Inc.     

Structural insight into the high reduction potentials observed for Fusobacterium nucleatum flavodoxin

Flavodoxins are small flavin mononucleotide (FMN)‐containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate‐binding loop (T/S‐X‐T‐G‐X‐T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The E_ox/sq (oxidized/semiquinone; ?43 mV) and E_sq/hq (semiquinone/hydroquinone; ?256 mV) are the highest recorded reduction potentials of known long‐chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70‐fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive E_ox/sq. The more electropositive E_sq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.     

Glu121-Lys319 salt bridge between catalytic and N-terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N

Escherichia coli aminopeptidase N (ePepN) belongs to the gluzincin family of M1 class metalloproteases that share a common primary structure with consensus zinc binding motif (HEXXH-(X18)-E) and an exopeptidase motif (GXMEN) in the active site. There is one amino acid, E121 in Domain I that blocks the extended active site grove of the thermolysin like catalytic domain (Domain II) limiting the substrate to S1 pocket. E121 forms a part of the S1 pocket, while making critical contact with the amino-terminus of the substrate. In addition, the carboxylate of E121 forms a salt bridge with K319 in Domain II. Both these residues are absolutely conserved in ePepN homologs. Analogous Glu-Asn pair in tricon interacting factor F3 (F3) and Gln-Asn pair in human leukotriene A(4) hydrolase (LTA(4) H) are also conserved in respective homologs. Mutation of either of these residues individually or together substantially reduced or entirely eliminated enzymatic activity. In addition, thermal denaturation studies suggest that the mutation at K319 destabilizes the protein as much as by 3.7 °C, while E121 mutants were insensitive. Crystal structure of E121Q mutant reveals that the enzyme is inactive due to the reduced S1 subsite volume. Together, data presented here suggests that ePepN, F3, and LTA(4) H homologs adopted a divergent evolution that includes E121-K319 or its analogous pairs, and these cannot be interchanged.     

The starch‐binding domain family CBM41—An in silico analysis of evolutionary relationships

Within the CAZy database, there are 81 carbohydrate‐binding module (CBM) families. A CBM represents a non‐catalytic domain in a modular arrangement of glycoside hydrolases (GHs). The present in silico study has been focused on starch‐binding domains from the family CBM41 that are usually part of pullulanases from the α‐amylase family GH13. Currently there are more than 1,600 sequences classified in the family CBM41, almost exclusively from Bacteria, and so a study was undertaken in an effort to divide the members into relevant groups (subfamilies) and also to contribute to the evolutionary picture of family CBM41. The CBM41 members adopt a β‐sandwich fold (～100 residues) with one carbohydrate‐binding site formed by the side‐chains of three aromatic residues that interact with carbohydrate. The family CBM41 can be divided into two basic subdivisions, distinguished from each other by a characteristic sequence pattern or motif of the three essential aromatics as follows: (i) “W‐W‐～10aa‐W” (the so‐called Streptococcus/Klebsiella‐type); and (ii) “W‐W‐～30aa‐W” (Thermotoga‐type). Based on our bioinformatics analysis it is clear that the first and second positions of the motif can be occupied by aromatic residues (Phe, Tyr, His) other than tryptophan, resulting in the existence of six different carbohydrate‐binding CBM41 groups, that reflect mostly differences in taxonomy, but which should retain the ability to bind an α‐glucan. In addition, three more groups have been proposed that, although lacking the crucial aromatic motif, could possibly employ other residues from remaining parts of their sequence for binding carbohydrate. Proteins 2017; 85:1480–1492. © 2017 Wiley Periodicals, Inc.     

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11

Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and K_m for pullulan of PulA are 750 U mg^?1 and 16.4 μmol L^?1, respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.     

Structure of dystrophia myotonica protein kinase

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled‐coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM‐8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled‐coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully‐ordered and correctly positioned αC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C‐terminal extension of the kinase domain is bound to the N‐terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C‐terminal extension compared to the closely related Rho‐associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM‐8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.     

Computational site‐directed mutagenesis studies of the role of the hydrophobic triad on substrate binding in cholesterol oxidase

Cholesterol oxidase (ChOx) is a flavoenzyme that oxidizes and isomerizes cholesterol (CHL) to form cholest‐4‐en‐3‐one. Molecular docking and molecular dynamics simulations were conducted to predict the binding interactions of CHL in the active site. Several key interactions (E361‐CHL, N485‐FAD, and H447‐CHL) were identified and which are likely to determine the correct positioning of CHL relative to flavin‐adenine dinucleotide (FAD). Binding of CHL also induced changes in key residues of the active site leading to the closure of the oxygen channel. A group of residues, Y107, F444, and Y446, known as the hydrophobic triad, are believed to affect the binding of CHL in the active site. Computational site‐directed mutagenesis of these residues revealed that their mutation affects the conformations of key residues in the active site, leading to non‐optimal binding of CHL and to changes in the structure of the oxygen channel, all of which are likely to reduce the catalytic efficiency of ChOx. Proteins 2017; 85:1645–1655. © 2017 Wiley Periodicals, Inc.     

Residues involved in the pore‐forming activity of the Clostridium perfringens iota toxin

Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334–359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic β‐strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the β‐pore‐forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic β‐strands affected pore formation, single‐channel conductance and ion selectivity (S339E‐S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Φ‐clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single‐channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Φ‐clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single‐channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E‐S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.     

Characterization of Mutant HIV-1 Integrase Carrying Amino Acid Changes in the Catalytic Domain

To gain insight into the importance of conserved residues in the core domain of HIV-1 IN, we performed site-directed mutagenesis of the full-length enzyme, overexpressed the mutant proteins in E. coli, purified and analyzed their 3-processing, integration and disintegration activities in vitro. Change of E152V in the DD(35)E motif abolished all detectable activities of IN. Alteration of two highly conserved residues, P145 and K156, by isoleucine, resulted in a substantial loss or completely abolished the three activities of the enzyme. Mutant P90D weakly reduced the 3-processing but severely affected the two other IN activities. Results obtained from double and triple mutations, P90D/P145I and P145I/F185K/C280S, clearly suggest a crucial role of P145 in the catalytic function of IN, whereas the mutants V150E, L158F and L172M had no detectable effect on any of the IN activities. Taken together, these results allowed us to conclude that all the conserved amino acids in the core domain of IN are not equally important for catalytic functions: like D64, D116 and E152, our data suggest that P90, P145 and K156 are also essential for all three enzymatic activities of HIV-1 IN in vitro, whereas V150, L158 and L172 appear to be less critical.     

Structural characterization of MepB from Staphylococcus aureus reveals homology to endonucleases

The MepRAB operon in Staphylococcus aureus has been identified to play a role in drug resistance. Although the functions of MepA and MepR are known, little information is available on the function of MepB. Here we report the X‐ray structure of MepB to 2.1 Å revealing its structural similarity to the PD‐(D/E)XK family of endonucleases. We further show that MepB binds DNA and RNA, with a higher affinity towards RNA and single stranded DNA than towards double stranded DNA. Notably, the PD‐(D/E)XK catalytic active site residues are not conserved in MepB. MepB's association with a drug resistance operon suggests that it plays a role in responding to antimicrobials. This role is likely carried out through MepB's interactions with nucleic acids.     

Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv

Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.