Tohru Yoshimura

CYSTEINE SULFINATE DESULFINASE, A NIFS-LIKE PROTEIN OF ESCHERICHIA COLI WITH SELENOCYSTEINE LYASE AND CYSTEINE DESULFURASE ACTIVITIES : GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF A NOVEL PYRIDOXAL ENZYME

Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. TheEscherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (M r43,238) and contains pyridoxal 5′-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms froml-cysteine, l-cystine,l-selenocysteine, and l-selenocystine to produce l-alanine. Because l-cysteine sulfinic acid was desulfinated to form l-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714–4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library : Identification of peptide substrates for tgase 2 and factor XIIIA

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPϕD(P), QxPϕ, and QxxϕDP; Factor XIIIa, QxxϕxWP (where x and ϕ represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS/IscU complex: Implications for the mechanism of iron-sulfur cluster assembly

IscS and IscU from Escherichia coli cooperate with each other in the biosynthesis of iron-sulfur clusters. IscS catalyzes the desulfurization of l-cysteine to produce l-alanine and sulfur. Cys-328 of IscS attacks the sulfur atom of l-cysteine, and the sulfane sulfur derived from l-cysteine binds to the Sγ atom of Cys-328. In the course of the cluster assembly, IscS and IscU form a covalent complex, and a sulfur atom derived from l-cysteine is transferred from IscS to IscU. The covalent complex is thought to be essential for the cluster biogenesis, but neither the nature of the bond connecting IscS and IscU nor the residues involved in the complex formation have been determined, which have thus far precluded the mechanistic analyses of the cluster assembly. We here report that a covalent bond is formed between Cys-328 of IscS and Cys-63 of IscU. The bond is a disulfide bond, not a polysulfide bond containing sulfane sulfur between the two cysteine residues. We also found that Cys-63 of IscU is essential for the IscU-mediated activation of IscS: IscU induced a six-fold increase in the cysteine desulfurase activity of IscS, whereas the IscU mutant with a serine substitution for Cys-63 had no effect on the activity. Based on these findings, we propose a mechanism for an early stage of iron-sulfur cluster assembly: the sulfur transfer from IscS to IscU is initiated by the attack of Cys-63 of IscU on the Sγ atom of Cys-328 of IscS that is bound to sulfane sulfur derived from l-cysteine.

Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5′-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. The enzyme also catalyzes the dehydration of d- and l-serine. Both reactions are enhanced by Mg·ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 Å resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with l-serine and d-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique “lysino-d-alanyl” residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme.

d-Amino acids in the brain: structure and function of pyridoxal phosphate-dependent amino acid racemases

d‐Serine serves as a co‐agonist of the N‐methyl d‐aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer’s disease and amyotrophic lateral sclerosis. d‐Serine is synthesized by a pyridoxal 5′‐phosphate (PLP)‐dependent serine racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP‐dependent amino acid racemase on the basis of its 3D structure. We compared the eukaryotic serine racemase with bacterial alanine racemase, the best‐studied enzyme among the PLP‐dependent amino acid racemases, and thus suggested a putative reaction mechanism for mammalian d‐serine synthesis.

Assembly of iron-sulfur clusters mediated by cysteine desulfurases, IscS, CsdB and CSD, from Escherichia coli

Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine. The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters. Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD. We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro. Since IscU, an iron-sulfur protein of E. coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU. By surface plasmon resonance analysis, we found that only IscS interacts with IscU. We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.

Alterations in d-amino acid concentrations and microbial community structures during the fermentation of red and white wines

Alterations in D-amino acid concentrations and microbial community structures during the fermentation of red and white wines were analyzed to clarify the relationship between the occurrence of d-amino acids and the actions of fermentative microorganisms. Relatives of Saccharomyces cerevisiae and Oenococcus oeni were detected in red wine samples, and relatives of S. cerevisiae, O. oeni, and Gluconacetobacter saccharivorans were detected in white wine samples. The S. cerevisiae relatives were detected throughout the fermentation process, whereas the O. oeni relatives were detected at the late stage of fermentation in both the red and white wine samples. The G. saccharivorans relative was detected in the early stage of fermentation. The amino acid analysis showed that D-alanine, D-glutamic acid, and D-lysine were present in both the red and white wine samples. The concentrations of these D-amino acids increased as the fermentation continued, especially from the malolactic fermentation stage to the end of the fermentation processes. These increases seem to be linked to the presence of O. oeni relatives.

New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

Mode of Action of a Germination-Specific Cortex-Lytic Enzyme, SleC, of Clostridium perfringens S40

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.

Geranylgeranyl reductase involved in the biosynthesis of archaeal membrane lipids in the hyperthermophilic archaeon Archaeoglobus fulgidus

Complete saturation of the geranylgeranyl groups of biosynthetic intermediates of archaeal membrane lipids is an important reaction that confers chemical stability on the lipids of archaea, which generally inhabit extreme conditions. An enzyme encoded by the AF0464 gene of a hyperthermophilic archaeon, Archaeoglobus fulgidus, which is a distant homologue of plant geranylgeranyl reductases and an A. fulgidus menaquinone‐specific prenyl reductase[Hemmi H, Yoshihiro T, Shibuya K, Nakayama T, & Nishino T (2005) J Bacteriol187, 1937–1944], was recombinantly expressed and purified, and its geranylgeranyl reductase activity was examined. The radio HPLC analysis indicated that the flavoenzyme, which binds FAD noncovalently, showed activity towards lipid‐biosynthetic intermediates containing one or two geranylgeranyl groups under anaerobic conditions. It showed a preference for 2,3‐di‐O‐geranylgeranylglyceryl phosphate over 3‐O‐geranylgeranylglyceryl phosphate and geranylgeranyl diphosphate in vitro, and did not reduce the prenyl group of respiratory quinones in Escherichia coli cells. The substrate specificity strongly suggests that the enzyme is involved in the biosynthesis of archaeal membrane lipids. GC‐MS analysis of the reaction product from 2,3‐di‐O‐geranylgeranylglyceryl phosphate proved that the substrate was converted to archaetidic acid (2,3‐di‐O‐phytanylglyceryl phosphate). The archaeal enzyme required sodium dithionite as the electron donor for activity in vitro, similarly to the menaquinone‐specific prenyl reductase from the same anaerobic archaeon. On the other hand, in the presence of NADPH (the preferred electron donor for plant homologues), the enzyme reaction did not proceed.