Yeast Ortholog of Peptidase Family M49: the Role of Invariant Glu(461) and Tyr(327)

Metallopeptidase family M49 is characterized by five conserved sequence regions and the unique motif HEXXGH with two histidines - ligands of the active-site zinc ion. The crystal structure of the yeast ortholog represents a prototype for the whole family. To investigate the role of two invariant amino acid residues, a Glu(461) of the zinc-binding motif, and a Tyr(327), 21 angstrom from the catalytic zinc center, mutational analysis of the yeast enzyme was performed. The substitution of Glu(461) to glutamine decreased k(cat) for the substrate hydrolysis almost by 10 000-fold. The replacement of Tyr(327) by Phe or Ala reduced the catalytic efficiency (k(cat)/K-m) by two orders of magnitude. The affinity for the heptapeptide valorphin was siginificantly lowered in all mutants, indicating the contribution of both Glu(461) and Tyr(327) in substrate binding. Taken together, the effect of mutating Glu(461) is consistent with this residue being essential in M49 peptidase catalysis

Reduced Flavin: NMR investigation of N(5)-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst

BACKGROUND: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. RESULTS: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the (15)N-NMR-signals of (15)N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the (15)N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the (15)N(5) signal and the proton exchange rates are little dependent on the buffer system used. CONCLUSION: The exchange rates allow an estimation of the pK(a )value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)(+)-H(2)) would be consistent with a role of N(5)-H as a base

Flavofun: Exploration of fungal flavoproteomes

Fungi produce a plethora of natural products exhibiting a fascinating diversity of chemical structures with an enormous potential for medical applications. Despite the importance of understanding the scope of natural products and their biosynthetic pathways, a systematic analysis of the involved enzymes has not been undertaken. In our previous studies, we examined the flavoprotein encoding gene pool in archaea, eubacteria, the yeast Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens. In the present survey, we have selected the model fungus Neurospora crassa as a starting point to investigate the flavoproteomes in the fungal kingdom. Our analysis showed that N. crassa harbors 201 flavoprotein-encoding genes amounting to 2% of the total protein-encoding genome. The majority of these flavoproteins (133) could be assigned to primary metabolism, termed the “core flavoproteome”, with the remainder of flavoproteins (68) serving in, as yet unidentified, reactions. The latter group of “accessory flavoproteins” is dominated by monooxygenases, berberine bridge enzyme-like enzymes, and glucose-methanol-choline-oxidoreductases. Although the exact biochemical role of most of these enzymes remains undetermined, we propose that they are involved in activities closely associated with fungi, such as the degradation of lignocellulose, the biosynthesis of natural products, and the detoxification of harmful compounds in the environment. Based on this assumption, we have analyzed the accessory flavoproteomes in the fungal kingdom using the MycoCosm database. This revealed large differences among fungal divisions, with Ascomycota, Basidiomycota, and Mucoromycota featuring the highest average number of genes encoding accessory flavoproteins. Moreover, a more detailed analysis showed a massive accumulation of accessory flavoproteins in Sordariomycetes, Agaricomycetes, and Glomeromycotina. In our view, this indicates that these fungal classes are proliferative producers of natural products and also interesting sources for flavoproteins with potentially useful catalytic properties in biocatalytic applications

The emerging role of dipeptidyl peptidase 3 in pathophysiology

Dipeptidyl peptidase 3 (DPP3), a zinc-dependent aminopeptidase, is a highly conserved enzyme among higher animals. The enzyme cleaves dipeptides from the N-terminus of tetra- to decapeptides, thereby taking part in activation as well as degradation of signalling peptides critical in physiological and pathological processes such as blood pressure regulation, nociception, inflammation and cancer. Besides its catalytic activity, DPP3 moonlights as a regulator of the cellular oxidative stress response pathway, e.g., the Keap1-Nrf2 mediated antioxidative response. The enzyme is also recognized as a key modulator of the renin-angiotensin system. Recently, DPP3 has been attracting growing attention within the scientific community, which has significantly augmented our knowledge of its physiological relevance. Herein, we review recent advances in our understanding of the structure and catalytic activity of DPP3, with a focus on attributing its molecular architecture and catalytic mechanism to its wide-ranging biological functions. We further highlight recent intriguing reports that implicate a broader role for DPP3 as a valuable biomarker in cardiovascular and renal pathologies and furthermore discuss its potential as a promising drug target

Direct expression of active spinach glycolate oxidase in Escherichia coli

Spinach glycolate oxidase (GAO) was expressed in Escherichia coli using the T7 RNA polymerase promotor. The enzyme accounts for approx. 1% of the soluble protein fraction and is expressed as a soluble and active enzyme. Comparison with GAO expressed in Saccharomyces cerevisiae (Macheroux, P., Massey, V., Thiele, D.J. and Volokita, M. (1991) Biochemistry 30, 4612-4619) showed that the GAO expressed in E. coli has identical physico-chemical features to the wild-type enzyme, but is expressed at a level approx. 15-fold higher than in the yeast system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29898/1/0000255.pd

Quinone reductase acts as a redox switch of the 20 S yeast proteasome

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102111/1/embr2008218.pd

Crystal structure of dipeptidyl peptidase III from the human gut symbiont Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron is a dominant member of the human intestinal microbiome. The genome of this anaerobe encodes more than 100 proteolytic enzymes, the majority of which have not been characterized. In the present study, we have produced and purified recombinant dipeptidyl peptidase III (DPP III) from B. thetaiotaomicron for the purposes of biochemical and structural investigations. DPP III is a cytosolic zinc-metallopeptidase of the M49 family, involved in protein metabolism. The biochemical results for B. thetaiotaomicron DPP III from our research showed both some similarities to, as well as certain differences from, previously characterised yeast and human DPP III. The 3D-structure of B. thetaiotaomicron DPP III was determined by X-ray crystallography and revealed a two-domain protein. The ligand-free structure (refined to 2.4 Å) was in the open conformation, while in the presence of the hydroxamate inhibitor Tyr-Phe-NHOH, the closed form (refined to 3.3 Å) was observed. Compared to the closed form, the two domains of the open form are rotated away from each other by about 28 degrees. A comparison of the crystal structure of B. thetaiotaomicron DPP III with that of the human and yeast enzymes revealed a similar overall fold. However, a significant difference with functional implications was discovered in the upper domain, farther away from the catalytic centre. In addition, our data indicate that large protein flexibility might be conserved in the M49 family

Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism

Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family