Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.3.1.8 (acyl-CoA dehydrogenase)
 785 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified by in vitro mutagenesis, and the sequence encoding the mature form of MCADH was introduced into an inducible expression plasmid. We observed synthesis of the protein in Escherichia coli cells transformed with this plasmid with measurable MCADH enzyme activity in cell extracts. Glutamic acid 376, which has been proposed by Powell and Thorpe (Powell, P. J., and Thorpe, J. (1988) Biochemistry 27, 8022-8028) as an essential residue and the proton-abstracting base at the active site of the enzyme, was mutated to glutamine. After expression in bacteria of this plasmid, the corresponding extracts show no detectable MCADH activity, although mutant MCADH-protein production was detected by protein immunoblots. The mature enzyme and the Gln376 mutant were purified to apparent homogeneity. The wild-type enzyme is a yellow protein due to the content of stoichiometric FAD and had a specific activity which is 50% of MCADH purified from pig kidney. The Gln376 mutant is devoid of activity (less than 0.02% that of wild type, expressed enzyme) and is green because of bound CoA persulfide. Properties of the mutant enzyme suggest that the Glu376----Gln change specifically affects substrate binding. These results prove that Glu376 plays an important role in the initial step of dehydrogenation catalysis.
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PMID:Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli. 197 May 66

We have used expression of human medium chain acyl-CoA dehydrogenase (MCAD) in Escherichia coli as a model system for dissecting the molecular effects of two mutations detected in patients with MCAD deficiency. We demonstrate that the R28C mutation predominantly affects polypeptide folding. The amounts of active R28C mutant enzyme produced could be modulated between undetectable to 100% of the wild-type control by manipulating the level of available chaperonins and the growth temperature. For the prevalent K304E mutation, however, the amounts of active mutant enzyme could be modulated only in a range from undetectable to approximately 50% of the wild-type, and the assembled mutant enzyme displayed a decreased thermal stability. Two artificially constructed mutants (K304Q and K304E/D346K) yielded clearly higher amounts of active MCAD enzyme than the K304E mutant but were also responsive to chaperonin co-overexpression and growth at low temperature. The thermal stability profile of the K304E/D346K double mutant was shifted to even lower temperatures than that of the K304E mutant, whereas that of the K304Q mutant was closely similar to the wild-type. Taken together, the results show that the K304E mutation affects (i) polypeptide folding due to elimination of the positively charged lysine and (ii) oligomer assembly and stability due to replacement of lysine 304 with the negatively charged glutamic acid.
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PMID:Effects of two mutations detected in medium chain acyl-CoA dehydrogenase (MCAD)-deficient patients on folding, oligomer assembly, and stability of MCAD enzyme. 773 Mar 33

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].
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PMID:Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active center base arrangement of long-chain Acyl-CoA dehydrogenase. 882 75

A gene from Mycobacterium tuberculosis coding for acyl-CoA dehydrogenase was cloned, overexpressed and characterized on the basis of enzyme activity with various chain length substrates. The results show that the protein is a medium chain acyl-CoA dehydrogenase (MCADH). The mycobacterium protein expressed appears to be unique, since by comparison, the active site glutamic acid of the protein does not lie in the same position as other well characterized MCADH, but in a position present in long chain and isovaleryl acyl-CoA dehydrogenases (LCADH and IVDH).
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PMID:Cloning and expression of an acyl-CoA dehydrogenase from Mycobacterium tuberculosis. 953 63