Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (Spanish National Research Council, CSIC), Madrid, Spain. Biochimica et Biophysica Acta
(Impact Factor: 4.66).
11/2011; 1824(2):339-49. DOI: 10.1016/j.bbapap.2011.10.016
The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.
Available from: jb.asm.org
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ABSTRACT: 3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7T. During investigation of a Tn5::mob-induced mutant defective in growth on 3,3′-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion
was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified
by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion
chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179
kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO32−). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 μmol min−1 mg−1, an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s−1 mM−1 for 3SP-CoA. However, AcdDPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily.
Comparison to other known desulfinases showed a comparably high catalytic efficiency of AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene
product that is responsible for the final desulfination step during catabolism of 3,3′-dithiodipropionate (DTDP), a sulfur-containing
precursor substrate for biosynthesis of polythioesters.
Available from: David Schleheck
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ABSTRACT: Hypotaurine (HT, 2-aminoethane-sulfinate) is known to be utilized by bacteria as a sole source of carbon, nitrogen and energy for growth, as is taurine (2-aminoethane-sulfonate), however, the corresponding HT-degradation pathway remained undefined. Genome-sequenced Paracoccus denitrificans PD1222 utilized HT (and taurine) quantitatively for heterotrophic growth and released the HT-sulfur as sulfite (and sulfate), and HT-nitrogen as ammonium. Enzyme assays with cell-free extracts suggested that a HT-inducible HT:pyruvate aminotransferase (Hpa) catalyzes the deamination of HT in an initial reaction step. Partial purification of the Hpa activity and peptide fingerprinting-mass spectrometry (PF-MS) identified the Hpa-candidate gene; it encoded an archetypal taurine:pyruvate aminotransferase (Tpa). The same gene-product was identified via differential PAGE and PF-MS, as well as the gene of a strongly HT-inducible aldehyde dehydrogenase (Adh). Both genes were overexpressed in E. coli. The overexpressed, purified Hpa/Tpa showed HT:pyruvate-aminotransferase activity. Alanine, acetaldehyde, and sulfite, were identified as the reaction products, but not sulfinoacetaldehyde; the reaction of Hpa/Tpa with taurine yielded sulfoacetaldehyde, which is stable. The overexpressed, purified Adh oxidized the acetaldehyde generated during the Hpa reaction to acetate in an NAD(+)-dependent reaction. Based on these results, the following degradation pathway for HT in strain PD1222 can be depicted. The identified aminotransferase converts HT to sulfinoacetaldehyde, which desulfinates spontaneously to acetaldehyde and sulfite; the inducible aldehyde dehydrogenase oxidizes acetaldehyde to yield acetate, which is metabolized, and sulfite, that is excreted.
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ABSTRACT: The sulfur atoms of sulfur-containing cofactors that are essential for numerous cellular functions in living organisms originate from L-cysteine via cysteine desulfurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologs. The existence of CSD orthologs in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulfur-trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulfur (S0)-reducing hyperthermophilic archaeon, encoding Tk-CSD, conferred a growth defect evident only in the absence of S0, and that growth can be restored by the addition of S0, but not sulfide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S0, but is necessary for iron-sulfur cluster biosynthesis in the absence of S0. Recombinant form of Tk-CSD expressed in Escherichia coli was obtained and it was found to catalyze the desulfuration of L-cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD-dependent iron-sulfur cluster biogenesis, which allows them to thrive outside solfataric environments.
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