NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

Abstract

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13 C-detection experiments. Biological contextAn orange-coloured protein (ORP), 117 residues long, was isolated from the sulphate reducing bacterium Desulfovibrio gigas grown in a lactate-sulphate medium under anaerobic conditions (Bursakov et al. 2004). Sequence homology search through sequenced or partially sequenced genomes shows that this gene is present in organisms that are synthrophic with methanogenic archaea, such as other species of Desulfovibrio (D.), as D. vulgaris Hildenborough and D. desulfuricans G20. However, the sequence identity between D. gigas ORP and the corresponding ORF in these organisms is 48% and 42%, respectively and no structural information is available for any of them. Moreover, a recent study using D. vulgaris Hildenborough and Methanosarcina barkeri has showed that the gene that codes for this protein is one of three putative encoding-genes whose expression is increased when the growth conditions are switched from syntrophic to sulphate reducing (Scholten et al. 2007), making the study of this protein more imperative in order to understand its physiological role.To our knowledge, the D. gigas ORP was the first of this family of proteins to be isolated in the holo-form. The metal cluster is composed of two molybdenum and one copper ions arranged in a novel mixed-metal sulphide cluster of the type [S 2 MoS 2 CuS 2 MoS 2 ] 3-, which is noncovalently bound to the polypeptide chain (Bursakov et al. 2004, George et al. 2000. However, the ORP expressed heterologously in Escherichia. coli does not contain the metal cluster, but attempts are currently being made in order to reconstitute the protein with a metal cluster.Solution structural studies have been performed in two other proteins of this family, the homologue from Thermotoga maritima (TM1290) and the one from Methanobacterium thermoautotrophicum (MTH1175) (Cort et al. 2000, Etezady-Esfarjani et al. 2004). Both proteins were obtained through the structural genomic approach: the ORFs with no known function were chosen from the complete genome of these bacteria, heterologously expressed and their structure determined in order to obtain functional insights. However, besides presenting low sequence identity with D. gigas ORP, 35% (TM1290) and 32% (MTH1175), no function has been identified so far for these proteins. Attending to the fact that these homology values are in the limit to apply homology modelling, we started our studies by the resonance assignment in order to obtain preliminary structural information about the apo-ORP from D. gigas. This is the starting point to further pursue the studies using the reconstituted holo-protein, in order to determine the binding site of the metal cluster and to obtain further structure-function insights on ...

The Anaerobe-Specific Orange Protein Complex of Desulfovibrio vulgaris Hildenborough Is Encoded by Two Divergent Operons Coregulated by σ 54 and a Cognate Transcriptional Regulator

“…ORP is a monomeric soluble protein that contains a novel metal sulfide (S 2 MoS 2 CuS 2 MoS 2 ) cluster noncovalently bound to the polypeptide chain (8,24,42). DVU2109 is a 487-residue protein composed of two domains: the N-terminal domain (residues 1 to 297) belongs to COG0489 (ATPases involved in chromosome partitioning), and the C-terminal domain (residues 375 to 480) belongs to COG1433.…”

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) fromDesulfovibrio gigas

“…It was obtained from a culture grown in a lactate-sulfate medium under anaerobic conditions. It has been proposed to be involved in a metabolic pathway that is activated when the growth conditions are switched from syntrophic to sulfatereducing (Pauleta et al, 2007). A structural homology search classifies ORP as a member of the nitrogenase accessory factor-like superfamily.…”

NiII‐ATCUN‐Catalyzed Tyrosine Nitration in the Presence of Nitrite and Sulfite

“…Furthermore, three new peaks (i = 7.672 ppm, ii = 7.381 ppm, and iii = 6.828 ppm) appeared.T he two highly affected His 3 protons, a and e, are shifted to ia nd ii, respectively,s upporting the notion of ad irect contact between this His 3 and Ni II . [22] Peak iii at 6.85 ppm (doublet) may correspondt othe H d /H e of Tyr 75 ,according to previousN MR data; [23] this means that the Ni II -ATCUN motif in Ni II -ATCUN-ORP interacts with the aromatic group of the Tyrr esidue. In the aliphatic region, only the alanine site resonances at 1.310 and 1.298 ppm are highly affected (not shown) in as imilarm anner to Cu-ATCUN-ORP, [20] but the Met signals at 1.987 and 1.890 ppm are not affected.…”

“…Fusion D. gigas protein, ATCUN-ORP,w as heterologous expressed in E. coli BL21(DE3) and purified in the apo-form as previously described. [23] Subsequently,1 m m Ni II Cl 2 was added to 1mm apo-ATCUN-ORP in 50 mm Tris-HCl, pH 7.5, and the mixture was incubated for 10 min on an ice bath (4 8C) to yield Ni II -ATCUN-ORP derivative. Formation of 3-NT in ATCUN-ORP was assessed by measuring the absorbance at 430 nm at pH 7.6.…”