Crystal structure of AibC, a reductase involved in alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus.

Isovaleryl coenzyme A (IV-CoA) performs a crucial role during development and fruiting-body formation in myxobacteria, which is reflected in the existence of a de novo biosynthetic pathway that is highly upregulated when leucine, the common precursor of IV-CoA, is limited. The final step in de novo IV-CoA biosynthesis is catalyzed by AibC, a medium-chain dehydrogenase/reductase. Here, the crystal structure of AibC from Myxococcus xanthus refined to 2.55 Šresolution is presented. The protein adopts two different conformations in the crystal lattice, which is a consequence of partial interaction with the purification tag. Based on this structure, it is suggested that AibC most probably uses a Zn(2+)-supported catalytic mechanism in which NADPH is preferred over NADH. Taken together, this study reveals structural details of the alternative IV-CoA-producing pathway in myxobacteria, which may serve as a base for further biotechnological research and biofuel production.

Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands.

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free IVD specific activity was 112.5 µmol porcine ETF min(-)(1) mg(-)(1), which was ∼20-fold higher than that of its CoA-persulfide bound form. The Km and catalytic efficiency (kcat/Km) for isovaleryl-CoA were 1.0 µM and 4.3 × 10(6) M(-1) s(-1) per monomer, respectively, and its Km for ETF was 2.0 µM. Anaerobic titration of isovaleryl-CoA into an IVD solution resulted in a stable blue complex with increased absorbance at 310 nm, decreased absorbance at 373 and 447 nm, and the appearance of the charge transfer complex band at 584 nm. The apparent dissociation constant (KDapp) determined spectrally for isovaleryl-CoA was 0.54 µM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropyl-acetyl-CoA, and ETF induced CD spectral changes at the 250-500 nm region while isobutyryl-CoA did not, suggesting conformational changes occur at the flavin ring that are ligand specific. Replacement of the IVD Trp166 with a Phe did not block IVD interaction with ETF, indicating that its indole ring is not essential for electron transfer to ETF. A twelve amino acid synthetic peptide that matches the sequence of the ETF docking peptide competitively inhibited the enzyme reaction when ETF was used as the electron acceptor with a Ki of 1.5 mM.

Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism.

Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors.

Identification of a novel IVD mutation in a consanguineous family with isovaleric acidemia.

Isovaleric acidemia (IVA) is a rare autosomal recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase encoded by IVD gene. In this case study we report the first Saudi IVA patients from a consanguineous family with a novel transversion (p.G362V) and briefly discuss likely phenotype-genotype correlation of the disease in the Saudi population. We explored the functional consequences of the mutation by using various bioinformatics prediction algorithms and discussed the likely mechanism of the disease caused by the mutation.

Clinical and molecular analysis of isovaleric acidemia patients in the United Arab Emirates reveals remarkable phenotypes and four novel mutations in the IVD gene.

Isovaleric acidemia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by deficiency of mitochondrial isovaleryl-CoA dehydrogenase (IVD). Accumulation of isovaleryl-CoA derivatives to toxic levels results in clinical symptoms of the disease. Here, we investigate the clinical and molecular features of Arab patients with IVA. Patients from five unrelated families were evaluated clinically and for defects in the IVD gene. Four novel mutations (p.F382fs, p.R392H, p.R395Q and p.E408K) have been identified with p.R395Q occurring in two families. In addition, molecular modeling of the identified missense mutations predicted their damaging effects on the protein and computational analysis of the p.F382fs mutation predicted the disruption of a 3' splicing site resulting in inactive or unstable gene product. Furthermore, we found an unusual case of a 17 years old female homozygous for the p.R392H mutation with no clinical symptoms. Our results illustrate a heterogeneous mutation spectrum and clinical presentation in the relatively small UAE population.

Biochemical characterization of isovaleryl-CoA dehydrogenase (LiuA) of Pseudomonas aeruginosa and the importance of liu genes fora functional catabolic pathway of methyl-branched compounds.

Growth of Pseudomonas aeruginosa on acyclic terpene alcohols (citronellol) and on other methyl-branched compounds such as leucine or isovalerate requires a functional leucine/isovalerate utilization (Liu) pathway. In this study, we investigated the liuABCDE gene cluster by insertion mutant analysis, heterologous expression of liuA in Escherichia coli and by biochemical characterization of purified LiuA protein. Mutants with insertion in any of the liu genes were unable to utilize acyclic terpenes or leucine/isovalerate and confirmed the importance of the liu genes for catabolism of methyl-branched compounds. An insertion mutant in liuA was complemented by a liuA copy in trans, indicating that possible polar downstream effects of the insertion are not essential for growth. LiuA purified from recombinant E. coli revealed acyl-CoA dehydrogenase activity with isovaleryl-CoA (KM 2.3 microM) and butyryl-CoA as substrates. Other acyl-CoA compounds such as isobutyryl-CoA, 3-hydroxybutyryl-CoA, octanoyl-CoA, citronellyl-CoA or 5-methyl-hex-4-enoyl-CoA were not utilized. Experimental evidence for expression and essential functions of other Liu proteins in metabolism of methyl-branched compounds is provided.

Functional analysis of acyl-CoA dehydrogenase catalytic residue mutants using surface plasmon resonance and circular dichroism.

The acyl-CoA dehydrogenases (ACDs) are a family of flavoenzymes involved in the metabolism of fatty acids and branched-chain amino acids. The ACDs share a similar structure and a common dehydrogenation mechanism in which a catalytic glutamate extracts a proton from an acyl-CoA substrate. The resulting charge-transfer complex subsequently passes electrons to electron-transferring flavoprotein (ETF). We previously generated catalytic residue mutants of human short-chain acyl-CoA dehydrogenase (SCAD) and isovaleryl-CoA dehydrogenase (IVD) that were difficult to characterize by traditional methods. In the present study, we developed a novel surface plasmon resonance-based assay to measure substrate binding to these mutants. Replacement of the catalytic glutamate in either SCAD or IVD with glycine resulted in a several-fold reduction in affinity for substrate. Circular dichroism studies substantiated our earlier findings that both SCAD E368G and IVD E254G are unable to form a charge-transfer complex with substrate/product. The CD spectra of IVD E254G also indicated a perturbation of the flavin environment, a finding supported by molecular modeling that predicted a shift in the conformation of a conserved tryptophan that lies in close proximity to the flavin. Lastly, competitive inhibition studies using the ETF fluorescence reduction assay suggested that SCAD E368G and IVD E254G do not effectively compete with the wild-type enzymes for the physiological electron acceptor ETF.