Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.
ABSTRACT 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.
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ABSTRACT: 3-Sulfinopropionyl-CoA desulfinase (Acd(DPN7)) is a new desulfinase that catalyzes the sulfur abstraction from 3-sulfinopropionyl-CoA (3SP-CoA) in the β-proteobacterium Advenella mimigardefordensis strain DPN7(T). 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 in an open reading frame with 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, Acd(DPN7) was heterologously expressed in E. coli BL21 (DE3) pLysS using pET23a::acd(DPN7). The purified protein is yellow and contains a non-covalently bound FAD cofactor as verified by HPLC-ESI-MS analyses. Size-exclusion chromatography revealed a native molecular weight 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 (SO(3)(2-)). Kinetic studies of Acd(DPN7) revealed a V(max) of 4.19 μmol min(-1) mg(-1), an apparent K(m) of 0.013 mM and a k(cat)/K(m) of 240.8 s(-1) mM(-1) for 3SP-CoA. However, Acd(DPN7) 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 the comparably high catalytic efficiency of Acd(DPN7) and indicated a novel reaction mechanism. Hence, Acd(DPN7) encodes a new desulfinase based on an acyl-CoA dehydrogenase (1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for the final desulfination step during catabolism of DTDP, a sulfur containing precursor substrate for biosynthesis of polythioesters.Journal of bacteriology 01/2013; DOI:10.1128/JB.02105-12 · 2.69 Impact Factor
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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.Journal of bacteriology 04/2013; DOI:10.1128/JB.00307-13 · 2.69 Impact Factor
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ABSTRACT: In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.PLoS ONE 01/2014; 9(7):e102139. DOI:10.1371/journal.pone.0102139 · 3.53 Impact Factor