Structural and spectroscopic characterization of a HdrA-like subunit from Hyphomicrobium denitrificans

Funding Information: We thank Laurenz Heidrich for help with statistical analyses. This work was supported by grant Da 351/8‐1 (to CD) from the Deutsche Forschungsgemeinschaft and Fundação para a Ciência e Tecnologia (Portugal) (grant PTDC/BIA‐BQM/29118 and R&D units MOSTMICRO‐ITQB (UIDB/04612/2020 and UIDP/04612/2020), and European Union's Horizon 2020 research and innovation program (grant agreement No 810856). Open access funding enabled and organized by Projekt DEAL. Publisher Copyright: © 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Many bacteria and archaea employ a novel pathway of sulfur oxidation involving an enzyme complex that is related to the heterodisulfide reductase (Hdr or HdrABC) of methanogens. As a first step in the biochemical characterization of Hdr-like proteins from sulfur oxidizers (sHdr), we structurally analyzed the recombinant sHdrA protein from the Alphaproteobacterium Hyphomicrobium denitrificans at 1.4 Å resolution. The sHdrA core structure is similar to that of methanogenic HdrA (mHdrA) which binds the electron-bifurcating flavin adenine dinucleotide (FAD), the heart of the HdrABC-[NiFe]-hydrogenase catalyzed reaction. Each sHdrA homodimer carries two FADs and two [4Fe–4S] clusters being linked by electron conductivity. Redox titrations monitored by electron paramagnetic resonance and visible spectroscopy revealed a redox potential between −203 and −188 mV for the [4Fe–4S] center. The potentials for the FADH•/FADH− and FAD/FADH• pairs reside between −174 and −156 mV and between −81 and −19 mV, respectively. The resulting stable semiquinone FADH• species already detectable in the visible and electron paramagnetic resonance spectra of the as-isolated state of sHdrA is incompatible with basic principles of flavin-based electron bifurcation such that the sHdr complex does not apply this new mode of energy coupling. The inverted one-electron FAD redox potentials of sHdr and mHdr are clearly reflected in the different FAD-polypeptide interactions. According to this finding and the assumption that the sHdr complex forms an asymmetric HdrAA′B1C1B2C2 hexamer, we tentatively propose a mechanism that links protein-bound sulfane oxidation to sulfite on HdrB1 with NAD+ reduction via lipoamide disulfide reduction on HdrB2. The FAD of HdrA thereby serves as an electron storage unit. Database: Structural data are available in PDB database under the accession number 6TJR.publishe

Biosynthetic Gene Cluster for the Cladoniamides, Bis-Indoles with a Rearranged Scaffold

The cladoniamides are bis-indole alkaloids isolated from Streptomyces uncialis, a lichen-associated actinomycete strain. The cladoniamides have an unusual, indenotryptoline structure rarely observed among bis-indole alkaloids. I report here the isolation, sequencing, and annotation of the cladoniamide biosynthetic gene cluster and compare it to the recently published gene cluster for BE-54017, a closely related indenotryptoline natural product. The cladoniamide gene cluster differs from the BE-54017 gene cluster in gene organization and in the absence of one N-methyltransferase gene but otherwise contains close homologs to all genes in the BE-54017 cluster. Both gene clusters encode enzymes needed for the construction of an indolocarbazole core, as well as flavin-dependent enzymes putatively involved in generating the indenotryptoline scaffold from an indolocarbazole. These two bis-indolic gene clusters exemplify the diversity of biosynthetic routes that begin from the oxidative dimerization of two molecules of l-tryptophan, highlight enzymes for further study, and provide new opportunities for combinatorial engineering

FACTORS AFFECTING MIGRATION FROM THE CROATIAN RURAL AREA

U radu se daju rezultati istraživanja migracija u seoskom području Republike Hrvatske. Cilj je istražiti čimbenike koji utječu na iseljavanje seoskog stanovništva Republike Hrvatske. Istraživanje je provedeno 2007. godine na uzorku od 914 ispitanika dobi od 24 do 45 godina u seoskom području Republike Hrvatske. Odabir naselja i ispitanika bio je slučajan. Provedeno istraživanje pokazuje da su najveće poteškoće života u hrvatskom seoskom području gospodarske naravi, manjak zaposlenja, slaba mogućnost izbora zanimanja i niža zarada u odnosu na zaposlenje u gradu. Petina ispitanika nije zadovoljna uvjetima seoskog života i namjerava se iseliti. To je zabrinjavajući pokazatelj budući da se radi o populaciji koja je u pravilu završila proces obrazovanja i većinom osnovala obitelj. Najviše mogućih iseljenika, što je bilo i za očekivati, je iz gospodarski nerazvijenih područja Republike Hrvatske. Daljnja depopulacija hrvatskog sela bila bi pogubna, a njene najveće posljedice bile bi: prevelika urbanizacija, posebice velikih gradova, daljnji neravnomjerni razvitak Republike Hrvatske te nedovoljno iskorištenje prostornog, proizvodnog i ljudskog potencijala. S obzirom na strateški cilj ulaska Republike Hrvatske u Europsku uniju, navedeno predstavlja bitno ograničenje njene uspješne prilagodbe europskoj ekonomskoj integraciji. Iseljavanje seoskog pučanstva može se spriječiti prvenstveno povećanjem zaposlenosti i dohotka te stvaranjem takve fizičke i društvene infrastrukture u seoskom području koja će bitno poboljšati životne uvjete seoskog pučanstva. Seoska područja, poglavito gospodarski nerazvijena, nemaju dovoljno vlastitih mogućnosti za ubrzanje razvoja odnosno za nužno smanjivanje razlika u kakvoći življenja prema gradskim područjima. Zbog toga je nužno da njihov razvojni proces više nego dosada potpomogne Država osmišljenim mjerama regionalnog razvoja, uz svekoliku potporu lokalne uprave i samouprave. U tome bi svoj znatan obol trebalo dati novo-osnovano Ministarstvo za regionalni razvoj.The paper presents results of the research study on migrations in rural areas of the Republic of Croatia. The aim was to determine factors influencing migrations of rural population in Croatia. The research was carried out in 2007 on 914 respondents from 25 to 45 years of age. The rural communities and respondents were selected on a random basis. The study results indicate that the major difficulties in rural life in Croatia are of economic nature: lack of employment opportunities, inadequate choice of profession and lower income in comparison with employment in urban areas. One fifth of the respondents is not satisfied with conditions of rural life and intends to leave villages. This is a very disturbing indicator, since it refers to population, which in general, has finished education and started a family. As we expect, the largest number of potential migrants comes from economically underdeveloped Croatian areas. Further depopulation of Croatian villages would have dramatic effects, and the worst consequences would be excessive urbanization, especially of large cities, further uneven development of the Republic of Croatia, and insufficient utilization of spatial, production and human resources. Since the strategic Croatian goal is to become a member of the European Union, this is a major obstacle to its successful adjustment to the European economic integration. The migration of rural population could be prevented primarily by increase in employment and income opportunities and creation of such physical and social infrastructure in rural areas that would considerably improve living conditions for rural population. The rural areas, particularly underdeveloped, have no adequate capacities for intensification of its development and diminishing differences in their quality of life compared to urban areas. Thus, the state support is increasingly required by introducing measures of regional development with complementary support of the local government. The newly founded Ministry of Regional Development is therefore inevitable in this process

CRYSTAL-STRUCTURES OF MUTANT PSEUDOMONAS-AERUGINOSA P-HYDROXYBENZOATE HYDROXYLASES - THE TYR201PHE, TYR385PHE, AND ASN300ASP VARIANTS

Structures of the mutant p-hydroxybenzoate hydroxylases, Tyr201Phe, Tyr385Phe, and Asn300Asp, each complexed with the substrate p-OHB have been determined by X-ray crystallography. Crystals of these three mutants of the Pseudomonas aeruginosa enzyme, which differs from the wild-type Pseudomonas fluorescens enzyme at two surface positions (228 and 249), were isomorphous with crystals of the wild-type P. fluorescens enzyme, allowing the mutant structures to be determined by model building and refinement, starting from the coordinates for the oxidized P. fluorescens PHBH-3,4-diOHB complex [Schreuder, H. A., van der Laan, J. M., Hol, W. G. J., & Drenth, J. (1988) J. Mol. Biol. 199, 637-648]. The R factors for the structures described here are: Tyr385Phe, 0.178 for data from 40.0 to 2.1 A; Tyr201Phe, 0.203 for data from 40.0 to 2.3 A; and Asn300Asp, 0.193 for data from 40.0 to 2.3 A. The functional effects of the Tyr201Phe and Tyr385Phe mutations, described earlier [Entsch, B., Palfey, B. A., Ballou, D. P., & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349], were rationalized with the assumption that the mutations perturbed the hydrogen-bonding interactions of the tyrosine residues but caused no other changes in the enzyme structure. In agreement with these assumptions, the positions of the substrate, the flavin, and the modified residues are not altered in the Tyr385Phe and Tyr201 Phe structures. In contrast, substitution of Asp for Asn at residue 300 has more profound effects on the enzyme structure. The side chain of Asp300 moves away from the flavin, disrupting the interactions of the carboxamide group with the flavin O(2) atom, and the ??-helix H10 that begins at residue 297 is displaced, altering its dipole interactions with the flavin ring. The functional consequences of these changes in the enzyme structure and of the introduction of the carboxyl group at 300 are described and discussed in the accompanying paper (Palfey et al., 1994b).close362

THE MOBILE FLAVIN OF 4-OH BENZOATE HYDROXYLASE

Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.close13212