Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site

Using the x-ray structure of the glycerol 3-phosphate transporter (GlpT), we devised a model for the distantly related oxalate transporter, OxlT. The model accommodates all earlier biochemical information on OxlT, including the idea that Lys-355 lies on the permeation pathway, and predicts that Lys-355 and a second positive center, Arg-272, comprise the binding site for divalent oxalate. Study of R272K, R272A, and R272Q derivatives verifies that Arg-272 is essential, and comparisons with GlpT show that both anion transporters bind substrates within equivalent domains. In 22 single-cysteine variants in TM7 and TM8, topology as marked by accessibility to Oregon green maleimide is predicted by the model, with similar concordance for 52 positions probed earlier. The model also reconciles cross-linking of a cysteine pair placed near the periplasmic ends of TM2 and TM7, and retrospective study of TM2 and TM11 confirms that positions supporting disulfide trapping lie at a helical interface. Our work describes a pathway to the modeling of OxlT and other transporters in the major facilitator superfamily and outlines simple experimental tests to evaluate such proposals.anion-binding ͉ disulfide trapping ͉ major facilitator superfamily ͉ membrane protein ͉ permeation pathway O xlT, the oxalate͞formate antiporter of Oxalobacter formigenes (1, 2), belongs to the major facilitator superfamily (MFS), a large and diverse collection encompassing 30-40% of known transporters and permeases (www.biology.ucsd.edu͞ϳmsaier͞ transport). The main biochemical mechanisms associated with transporters (uniport, antiport, and symport) may be found within the MFS, whose individual members display a broad catalog of substrate specificity, including simple sugars and amino acids, intermediary metabolites, and even neurotransmitters (3). All members of the MFS share an architectural theme in which a central loop connects two groups of (typically) six transmembrane ␣-helices. Moreover, the superfamily as a whole is characterized by a short motif (GXXXDK͞R) at the cytoplasmic ends of TM2 and TM8 (3, 4), suggesting that these two six-helix clusters derived from a common ancestor; indeed, at times one finds a clear sequence homology between the N-and C-terminal domains (4, 5).Insight into the structure of MFS transporters is presently limited. Helix organization, symmetry, and connectivity were established first for OxlT, in work based on a low-resolution (6.5 Å) structure obtained by electron crystallography (6, 7). Subsequently, higher resolution (3.2-3.5 Å) was achieved by x-ray crystallography of two other MFS members from Escherichia coli, the H ϩ ͞lactose symporter (LacY) and the phosphate͞glycerol 3-phosphate antiporter (GlpT) (8, 9). These latter achievements have prompted several recent attempts to use LacY or GlpT as structural templates for models of other systems (10-13). With this in mind and to provide a detailed perspective to guide further work, we selected the GlpT structure as a template for derivation of a homology model of OxlT, ...

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“…3). Importantly, such dimensions are consistent with those found for ligand association in oxalate binding proteins of known structure (27,28).…”

Section: Discussionsupporting

confidence: 84%

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Yang

Wang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…4A). The residues proposed by Walsh et al (42) to constitute the catalytic triad of the enzyme (R96, E99, and S122 in E. coli nomenclature) are also conserved in the CynS protein from P. pseudoalcaligenes (Fig. 4A).…”

Section: Fig 5 Tolerance Of the Wild Type (White Bars) And Cynsmentioning

confidence: 83%

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation

Luque-Almagro

Huertas

Sáez

et al. 2008

Appl Environ Microbiol

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Cyanase catalyzes the decomposition of cyanate into CO 2 and ammonium, with carbamate as an unstable intermediate. The cyanase of Pseudomonas pseudoalcaligenes CECT5344 was negatively regulated by ammonium and positively regulated by cyanate, cyanide, and some cyanometallic complexes. Cyanase activity was not detected in cell extracts from cells grown with ammonium, even in the presence of cyanate. Nevertheless, a low level of cyanase activity was detected in nitrogen-starved cells. The cyn gene cluster of P. pseudoalcaligenes CECT5344 was cloned and analyzed. The cynA, cynB, and cynD genes encode an ABC-type transporter, the cynS gene codes for the cyanase, and the cynF gene encodes a novel 54 -dependent transcriptional regulator which is not present in other bacterial cyn gene clusters. The CynS protein was expressed in Escherichia coli and purified by following a simple and rapid protocol. The P. pseudoalcaligenes cyanase showed an optimal pH of 8.5°C and a temperature of 65°C. An insertion mutation was generated in the cynS gene. The resulting mutant was unable to use cyanate as the sole nitrogen source but showed the same resistance to cyanate as the wild-type strain. These results, in conjunction with the induction pattern of the enzymatic activity, suggest that the enzyme has an assimilatory function. Although the induction of cyanase activity in cyanide-degrading cells suggests that some cyanate may be generated from cyanide, the cynS mutant was not affected in its ability to degrade cyanide, which unambiguously indicates that cyanate is not a central metabolite in cyanide assimilation.

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“…In response to cyanate addition, transcription was induced as a polycistronic message of cynS, together with a cyanate transporter gene (1). Twenty years later, the protein structure and subunit organization of E. coli CynS were determined at 1.65-Å resolution (38). The cyanase monomer was found to be composed of two domains: an N-terminal domain with similarity to the DNA-binding ␣-helix bundle motif and an "open-fold" C-terminal domain with no structural homology to other proteins.…”

mentioning

confidence: 99%

Characterization of Cyanate Metabolism in Marine Synechococcus and Prochlorococcus spp

Kamennaya

Post

2011

Appl Environ Microbiol

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Cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, occupying a key position at the base of marine food webs. The cynS gene that encodes cyanase was identified among bacterial, fungal, and plant sequences in public databases, and the gene was particularly prevalent among cyanobacteria, including numerous Prochlorococcus and Synechococcus strains. Phylogenetic analysis of cynS sequences retrieved from the Global Ocean Survey database identified >60% as belonging to unicellular marine cyanobacteria, suggesting an important role for cyanase in their nitrogen metabolism. We demonstrate here that marine cyanobacteria have a functionally active cyanase, the transcriptional regulation of which varies among strains and reflects the genomic context of cynS. In Prochlorococcus sp. strain MED4, cynS was presumably transcribed as part of the cynABDS operon, implying cyanase involvement in cyanate utilization. In Synechococcus sp. strain WH8102, expression was not related to nitrogen stress responses and here cyanase presumably serves in the detoxification of cyanate resulting from intracellular urea and/or carbamoyl phosphate decomposition. Lastly, we report on a cyanase activity encoded by cynH, a novel gene found in marine cyanobacteria only. The presence of dual cyanase genes in the genomes of seven marine Synechococcus strains and their respective roles in nitrogen metabolism remain to be clarified.

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Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site

Cited by 52 publications

References 59 publications

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation

Characterization of Cyanate Metabolism in Marine Synechococcus and Prochlorococcus spp

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