Structure and Substrate Specificity of the Pyrococcal Coenzyme A Disulfide Reductases/Polysulfide Reductases (CoADR/Psr): Implications for S0-Based Respiration and a Sulfur-Dependent Antioxidant System inPyrococcus

Biochemistry ◽  
2013 ◽  
Vol 52 (16) ◽  
pp. 2764-2773 ◽  
Author(s):  
Sanna Herwald ◽  
Albert Y. Liu ◽  
Brian E. Zhu ◽  
Kevin W. Sea ◽  
Karlo M. Lopez ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Antioxidant System ◽  
Coenzyme A ◽  
Disulfide Reductases

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

Computational Studies on the Substrate Specificity of an Acyltransferase Domain from Salinomycin Polyketide Synthase

2021 ◽  
Author(s):  
Huining Ji ◽  
Ting Shi ◽  
Lei Liu ◽  
Fa Zhang ◽  
Wentao Tao ◽  
...  
Keyword(s):  
Natural Products ◽  
Substrate Specificity ◽  
Polyketide Synthase ◽  
Coenzyme A ◽  
Biological Activities ◽  
Polyketide Synthases ◽  
Short Chain ◽  
Computational Studies ◽  
Chemical Structures ◽  
Coa Thioesters

Polyketides are a large group of natural products with diverse chemical structures and biological activities. They are biosynthesized by modular polyketide synthases (PKSs) from coenzyme A (CoA) thioesters of short-chain...

scholarly journals Substrate Specificity of the 3-Methylcrotonyl Coenzyme A (CoA) and Geranyl-CoA Carboxylases from Pseudomonas aeruginosa

2008 ◽  
Vol 190 (14) ◽  
pp. 4888-4893 ◽  
Author(s):  
J. A. Aguilar ◽  
C. Díaz-Pérez ◽  
A. L. Díaz-Pérez ◽  
J. S. Rodríguez-Zavala ◽  
B. J. Nikolau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Recombinant Proteins ◽  
Coenzyme A ◽  
Affinity Constant ◽  
Kinetic Behavior ◽  
Sigmoidal Kinetics ◽  
Leucine Catabolism ◽  
Temperature Optima

ABSTRACT Biotin-containing 3-methylcrotonyl coenzyme A (MC-CoA) carboxylase (MCCase) and geranyl-CoA (G-CoA) carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37°C. The apparent K 0.5 (affinity constant for non-Michaelis-Menten kinetics behavior) values of MCCase for MC-CoA, ATP, and bicarbonate were 9.8 μM, 13 μM, and 0.8 μM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate G-CoA. In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed sigmoidal kinetic behavior for G-CoA and bicarbonate but showed Michaelis-Menten kinetics for MC-CoA and the cosubstrate ATP. The apparent K 0.5 values of GCCase were 8.8 μM and 1.2 μM for G-CoA and bicarbonate, respectively, and the apparent Km values of GCCase were 10 μM for ATP and 14 μM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both the MCCase and GCCase enzymes play important roles in the leucine and acyclic terpene catabolic pathways.

scholarly journals Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase

2008 ◽  
Vol 190 (7) ◽  
pp. 2556-2564 ◽  
Author(s):  
Cory G. Toyota ◽  
Catrine L. Berthold ◽  
Arnaud Gruez ◽  
Stefán Jónsson ◽  
Ylva Lindqvist ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Coenzyme A ◽  
Substrate Inhibition ◽  
Kinetic Behavior ◽  
Oxalobacter Formigenes ◽  
Structural Genomic ◽  
E Coli ◽  
Coa Transferase ◽  
Acidic Conditions

ABSTRACT The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.

Cloning of a cDNA for Short/Branched Chain Acyl-Coenzyme A Dehydrogenase from Rat and Characterization of Its Tissue Expression and Substrate Specificity

1996 ◽  
Vol 331 (1) ◽  
pp. 127-133 ◽  
Author(s):  
Jan Willard ◽  
Caroline Vicanek ◽  
Kevin P. Battaile ◽  
Paul P. Van Veldhoven ◽  
Abdul H. Fauq ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Coenzyme A ◽  
Tissue Expression ◽  
Chain Acyl ◽  
Branched Chain ◽  
Acyl Coenzyme A
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