Mutational analysis and characterization of nocardicin C-9' epimerase

The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806 was recently identified. Nocardicin A is the most potent of a series of monocyclic beta-lactam antibiotics produced by this organism. Its activity has been attributed to a syn-configured oxime moiety and a d-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework. Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5'-phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases. Insertional mutagenesis of nocJ abolished nocardicin A production, while the l-homoseryl isomer, isonocardicin A, was still observed. Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant. Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9' epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C. NocJ is the first member of a new class of beta-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.     

Analysis of the codon usage of the cephamycin C producer Nocardia lactamdurans

Abstract The G + C content in a sequenced region of 27 kb of the Nocardia lactamdurans genome is 70.4 and 70.6% in the 14 characterized ORFs, showing an extreme average G + C content (94.9%) in the third codon position. The codon usage parameters of the N. lactamdurans genes studied are closely related and depart weakly from the values of other species of the genus Nocardia . The homologies and differences in the codon usage between N. lactamdurans and Streptomyces sp. or other high-G + C Gram-positive genera are analysed.     

Electron microscopic study of active and inactive variants of Proactinomyces (Nocardia) mediterranei, a producer of rifamycin B

Four mutants of Proactinomyces (Nocardia) mediterranei, an organism producing rifamycin B were subjected to electron microscopic investigation. The mutants differed in the biosynthetic activity. The investigation showed correlation between the length of the hyphal fragments of the over-substrate mycelium and the level of the organism activity. The study of the fine structure of the mycelium revealed changes in the structure of the cell wall, character of the nuclear material and hyphae vacuolization.     

Mutational analysis of a baculovirus major late promoter

We have used a linker-scan mutation strategy to analyze P_cap99, the proximal promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) gene encoding the major capsid protein. A series of recombinant viruses expressing the cat reporter gene under the control of selected mutants of this promoter was constructed. Only mutations that altered bases within a region extending from 8 bp upstream to 6 bp downstream from a TAAG sequence had a significant effect on expression from the late gene promoter. A synthetic promoter consisting of only these 18 bp (P_capmin) was sufficient to direct late expression. Aside from this small region surrounding the TAAG, no evidence for distinct late activating or repressing sequence elements was obtained. Experiments comparing and combining late and very late gene promoter sequences suggest that late expression is intrinsically determined by the presence and immediate context of a TAAG sequence and that very late expression [as previously shown in Ooi et al. J. Mol. Biol. 210 (1989) 721–736] results from additional modulation of TAAG-dependent expression by downstream promoter elements placed in an appropriate context. A compact combination promoter (95 bp), constructed by fusing P_capmin to linker-modified very late polyhedrin promoter, directs strong expression at late and very late times post-infection.     

Mutational analysis of a ras catalytic domain.

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.     

Mutational and biochemical analysis of dopamine in dystonia

The dystonias are a group of serious movement disorders characterized by involuntary muscle spasms of different parts of the body. We recently proposed that hypofunction of dopamine D₂ receptor-mediated inhibition of the indirect output pathway of the basal ganglia can result in dystonia. In this review, we discuss the results of a variety of genetic and biochemical studies in light of this hypothesis. Several forms of early-onset dystonia show distinct autosomal dominant, recessive, or X-linked genetic transmission patterns. Late onset forms of dystonia, though not showing clear Mendelian transmission patterns, also appear to be highly familial. Recently, several genetic-linkage locations have been identified for early-onset dystonia and for two of these loci, mutations decreasing dopamine synthesis have been demonstrated. Biochemical studies of monkeys and man also demonstrate that several types of dystonia occur in a dopamine-deficiency state. Similarly, mice strains developed to be deficient in several dopamine-pathway components have motor abnormalities consistent with dystonia. Hypofunction of the dopamine D₂ receptormediated inhibition of the indirect output pathway of the putamen may be a common feature of many of these heritable and secondary dystonic syndromes.     

Mutational analysis of the S21 pinholin

Lambdoid phage 21 has the prototype pinholin‐SAR endolysin lysis system, which is widely distributed among phages. Its prototype pinholin, S²¹68, triggers at an allele‐specific time to form small, heptameric lesions, or pinholes, in the cytoplasmic membrane, thus initiating lysis. S²¹68 has two transmembrane domains, TMD1 and TMD2. Only TMD2 is required for the formation of pinholes, whereas TMD1 acts as an inhibitor of TMD2 and must be externalized to the periplasm in the lytic pathway. Previously we provided evidence that S²¹68 first accumulates as inactive dimers with both transmembrane domains embedded in the bilayer. Here we analyse an extensive collection of S²¹ mutants to identify residues and domains critical to the function and regulation of the pinholin. Evidence is presented indicating that, within the inactive dimer, TMD1 acts in trans as an inhibitor of the lethal function of TMD2. A wide range of phenotypes, from absolute lysis defectives to accelerated lysis triggering, are observed for mutations mapping to each topological domain. The pattern of phenotypes allows the generation of a model for the structure of the inactive dimer. The model identifies the faces of the two transmembrane domains involved in intramolecular and intermolecular interactions, as well as interaction with the lipid.     

Mutational analysis of a mammalian reovirus mRNA capping enzyme

The amino-terminal 42-kDa region of the 144-kDa mammalian reovirus lambda 2 protein is a guanylyltransferase. It catalyzes the transfer of GMP from GTP to the 5' end of 5' -diphosphorylated mRNA via a phosphoamide with Lys-190. This amino acid is located at the base of a deep cleft. Based on sequence comparisons, the Kx[V/L/I]S motif is present in all known and proposed guanylyltransferases of the family Reoviridae. The requirement for this conserved sequence and other regions of the enzyme was analyzed by site-directed mutagenesis. Based on the enzymatic activity of the mutants, Lys-190 and Asp-191 are the only amino acids of the (190)KDLS sequence that are necessary for enzymatic activity. Since Asp-191 has its side chain oriented away from the cleft, most likely it plays an indirect role in forming a functional guanylyltransferase.     

Mutational analysis of a sequence-specific ssDNA binding lupus autoantibody

11F8 is a murine anti-ssDNA monoclonal autoantibody isolated from a lupus prone autoimmune mouse. This mAb binds sequence specifically, and prior studies have defined the thermodynamic and kinetic basis for sequence-specific recognition of ssDNA (Ackroyd, P. C., et al. (2001) Biochemistry 40, 2911-2922; Beckingham, J. A. and Glick, G. D. (2001) Bioorg. Med. Chem. 9, 2243-2252). Here we present experiments designed to identify the residues on 11F8 that mediate sequence-specific, noncognate, and nonspecific recognition of ssDNA and their contribution to the overall binding thermodynamics. Site-directed mutagenesis of an 11F8 single-chain construct reveals that six residues within the complementarity determining regions of 11F8 account for ca. 80% of the binding free energy and that there is little cooperativity between these residues. Germline-encoded aromatic and hydrophobic side chains provides the basis for nonspecific recognition of single-stranded thymine nucleobases. Sequence-specific recognition is controlled by a tyrosine in the heavy chain along with a somatically mutated arginine residue. Our data show that the manner in which 11F8 achieves sequence-specific recognition more closely resembles RNA-binding proteins such as U1A than other types of nucleic acid binding proteins. In addition, comparing the primary sequence of 11F8 with clonally related antibodies that differ by less than five amino acids suggests that somatic mutations which confer sequence specificity may be a feature that distinguishes glomerulotrophic pathogenic anti-DNA from those that are benign.     

Mutational analysis of ABC proteins

The 49 human members of the ATP-binding cassette (ABC) family of proteins are involved in a wide range of activities such as active transport of compounds across membranes, extraction of compounds out of membranes, functioning as ion channels, or regulators of channel activity. Mutations and/or overexpression of many of the proteins can have adverse effects on health. A goal in the study of ABC proteins is to understand their mechanisms of action. This review will focus on the mutational approaches that have been used to study the structure and mechanisms of some ABC proteins.     

Mutational analysis of the disulfide catalysts DsbA and DsbB

In prokaryotes, disulfides are generated by the DsbA-DsbB system. DsbB functions to generate disulfides by quinone reduction. These disulfides are passed to the DsbA protein and then to folding proteins. To investigate the DsbA-DsbB catalytic system, we performed an in vivo selection for chromosomal dsbA and dsbB mutants. We rediscovered many residues previously shown to be important for the activity of these proteins. In addition, we obtained one novel DsbA mutant (M153R) and four novel DsbB mutants (L43P, H91Y, R133C, and L146R). We also mutated residues that are highly conserved within the DsbB family in an effort to identify residues important for DsbB function. We found classes of mutants that specifically affect the apparent K(m) of DsbB for either DsbA or quinones, suggesting that quinone and DsbA may interact with different regions of the DsbB protein. Our results are consistent with the interpretation that the residues Q33 and Y46 of DsbB interact with DsbA, Q95 and R48 interact with quinones, and that residue M153 of DsbA interacts with DsbB. All of these interactions could be due to direct amino acid interactions or could be indirect through, for instance, their effect on protein structure. In addition, we find that the DsbB H91Y mutant severely affects the k(cat) of the reaction between DsbA and DsbB and that the DsbB L43P mutant is inactive, suggesting that both L43 and H91 are important for the activity of DsbB. These experiments help to better define the residues important for the function of these two protein-folding catalysts.     

Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', and C3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism.     

Mutational analysis of chilling tolerance in plants

A mutational approach was taken to identify genes required for low-temperature growth of the chilling-tolerant plant Arabidopsis thaliana. The screen identified mutants that were specifically compromised in their ability to grow at 5°C but were indistinguishable from wild type when grown at 22°C. The populations screened were mutated either by ethyl methanesulphonate or by T-DNA insertion. In both cases symptoms at 5°C included chlorosis, reduced growth, necrosis and death. This diversity of phenotypes demonstrates roles for chilling-tolerance responses in such diverse processes as organdie biogenesis, cell metabolism and cell and organ development. Co-segregation analysis on the first five mutants isolated from the T-DNA lines indicated that in three of them, pfc1, pfc2 and sop1, the chilling phenotype is the result of T-DNA insertion in a gene required for chilling tolerance rather than the creation of a temperature-conditional mutation in an essential housekeeping gene. This identification of T-DNA tagged alleles will facilitate cloning of the PFC1, PFC2 and SOP1 loci and allow for the biochemical and molecular genetic characterization of these chilling-tolerance genes and the proteins that they encode.     

Mutational analysis and a structural model of methyl-directed restriction enzyme Mrr

The Mrr protein of Escherichia coli K12 is a cryptic Type IV restriction endonuclease whose activity appears to be triggered by high pressure stress. In this report we used high pressure to isolate and analyze several Mrr mutants, and generated a new structural model of the Mrr protein. The activity of a number of spontaneous and strategically constructed Mrr mutants is discussed in the light of this model, providing a first insight into the structure-function relationships of the Mrr enzyme.     

Mutational analysis of blue-light sensing in Arabidopsis

Blue light regulates many physiological and developmental processes in higher plants through the action of multiple photosensory systems. The analysis of photomorphogenic mutants is leading to a better understanding of how the different photosensory systems mediate the wide range of responses observed in blue light. A review of the current literature on photomorphogenic mutants makes it apparent that redundancies exist in photoreceptor function. For example, many blue-light responses that have been shown to be regulated by a blue-light photosensory system are also under phytochrome control. The study of various light-response mutants suggests that a complex sensory network regulates light-mediated responses. This article attempts to piece together information regarding the sensory systems responsible for blue-light-regulated responses.