Improved Stability of Baeyer–Villiger Mono-Oxygenase from Pseudomonas fluorescens by Substitution of Cysteine Residues

Baeyer–Villiger mono-oxygenases (BVMOs) are enzymes that could insert an oxygen into carbon skeletons adjacent to carbonyl groups to form lactone or ester. Recently, a new method of sebacic acid production from oleic acid based on Pseudomonas fluorescens DSM 50106 (PfBVMO) catalysis was reported. However, the instable property of PfBVMO limited the further investigation and application of this enzyme. In this study, we aimed to improve the stability of PfBVMO by sitedirected mutagenesis. The sequence of PfBVMO indicates probable factors from the susceptible oxidizing cysteine residue leading to the instability of PfBVMO. Activity and stability of mutants and the wild-type were characterized to investigate the correlation of cysteine residues and activity. The mutant enzyme with entire cysteine residues replaced by serine residues, showed significantly higher stability than the wild type. The result would facilitate further work on the enzyme modification and crystal structure determination. Meanwhile, the results of this study provided potential for other enzyme stability evolution.

Identification of an inactivating cleavage site for α-thrombin on the heavy chain of factor Va

SummaryPrevious studies of factor (F)Va inactivation on human umbilical vein endothelial cells have shown that α-thrombin cleaves the heavy chain near the COOH-terminus to produce a Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 8,000 peptide containing the rest of the molecule. The α-thrombin cleavage appeared to occur between amino acid residues 586 and 654 of FV. This region contains a consensus sequence for α-thrombin cleavage located at residues 640–644 (S-S-P-R-S). To test the hypothesis that α-thrombin cleaves the FVa heavy chain at Arg643 and to evaluate the functional importance of this cleavage for FVa inactivation, sitedirected mutagenesis was used to create recombinant FV molecules with mutations R643→Q (FVR643Q) and R643→A (FVR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with α-thrombin. Under similar experimental conditions, appearance of the Mr 97,000 heavy chain fragment in the plasma and wild-type FVa molecules correlated with partial loss of cofactor activity, while following extended incubation of FVR643Q and FVR643A with α-thrombin no cleavage of the heavy chain at Arg643 was detected and no presence of the Mr 97,000 heavy-chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant FVa molecules. Our data demonstrate that cleavage of FVa at Arg643 by α-thrombin results in a partially inactive cofactor molecule and provides for an activated protein C (APC)-independent anticoagulant effect of α-thrombin.

Zinc Ligands in an Astacin Family Metalloprotease Meprin A

A conserved tyrosine residue in the astacin family of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Sitedirected mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin α was used to test the hypothesis that this residue is essential for zinc binding and enzymatic activity. In addition, another proposed zinc binding ligand, H167, in the conserved (HEXXH) zinc binding motif of the meprin α protease domain was replaced by an alanine residue. Both mutants were expressed and secreted with the same subunit mass as wild type (90 kDa). The Y226F mutant retained the capacity to oligomerize to higher covalently and noncovalentlylinked oligomers as the wild type, whereas H167A was predominantly a monomer. The kcat/Km for Y226F against a fluorgenic bradykinin substrate analog was approximately 15% of the wild type, while the H167A mutant had no detectable activity. Both Y226F and H167A were more susceptible to extensive degradation by trypsin compared with the wildtype protein. The zinc content in the wildtype and Y226F mutant proteins were similar, one molecule of zinc per subunit. The results indicate that Y226 is not essential for zinc binding, but Y226 and H167 are essential for full enzymatic activity and stability of the metalloproteinase.

Functional Analysis of the Escherichia coli Molybdopterin Cofactor Biosynthesis Protein MoeA by Site-Directed Mutagenesis

Five moeA mutants were generated by replacing some conserved amino acids of MoeA by sitedirected mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Momolybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterinderived dephospho FormA as compared to bacteria transformed with wildtype moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.

Identification of a Gephyrin-Binding Motif in the GDP/GTP Exchange Factor Collybistin

The brainspecific GDP/GTP exchange factor collybistin interacts with the receptoranchoring protein gephyrin and activates the Rholike GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with heterooligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pulldown assays and in the yeast twohybrid system. Sitedirected mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyRβsubunit binding motif. Our data are consistent with collybistingephyrin interactions occuring during inhibitory postsynaptic membrane formation.

Erratum

Import of DNA from the cytoplasm into the mitochondrial matrix is an obligatory step for an in organello sitedirected mutagenesis or gene therapy approach on mitochondrial DNA diseases. In this context, we have developed an artificial DNA translocation vector that is composed of the mitochondrial signal peptide of the ornithine transcarbamylase (OTC) and a DNA moiety. While this vector is capable of directing attached passenger molecules to the mitochondrial matrix, the recognition of this artificial molecule by the endogenous mitochondrial signal peptide processing machinery as well as the cleavage of the peptide plays a pivotal role in the release of the attached DNA. To study the proteolytic processing of the artificial vector, various signal peptideDNAconjugates were treated with purified mitochondrial intermediate peptidase. When the leader peptide is directly linked to the DNA moiety without an intervening spacer, MIP processing is prevented. Cleavage of the peptide can be restored, however, when the first ten amino acid residues of the mature part of OTC are appended at the carboxyterminal end of the signal peptide. Our results show that artificial peptideDNAconjugates are recognized by the mitochondrial proteolytic machinery, and therefore an interference of the peptide with the DNA function can be excluded.

Permutation of the Active Site Motif of Tryparedoxin 2

Tryparedoxins (TXN) are thioredoxinrelated proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothionedependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, [40]WCPPCR[45], of Crithidia fasciculata was mutated by sitedirected mutagenesis and eight corresponding muteins were expressed in E. coli as terminally Histagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarily the reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.