Medium-Long-Chain Chimeric Human Acyl-CoA Dehydrogenase:  Medium-Chain Enzyme with the Active Center Base Arrangement of Long-Chain Acyl-CoA Dehydrogenase

Abstract

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate R-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 isovalerylCoA 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 ∼20% of the activity of MCADH and ∼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].Acyl-CoA dehydrogenases are a class of flavoproteins that catalyze the desaturation of acyl-CoA substrates. Four known members are involved in mammalian mitochondrial -oxidation of fatty acids (short-, medium-, long-, and very long chain acyl-CoA dehydrogenases), and three (isovaleryl-, isobutyryl-and glutaryl-CoA dehydrogenases) are involved in the degradation of amino acids. In addition microsomal or peroxisomal acyl-CoA oxidases and a variety of related enzymes of bacterial or plant origin can be viewed as sharing the capacity to catalyze the seemingly identical chemical process, the substrate dehydrogenation at the positions R, .

Inversion of stereospecificity of vanillyl-alcohol oxidase

“…Nevertheless, the above data show that subtle variations in active site topology determine the outcome of the enzyme stereospecificity and reinforce the idea that inversion of enantioselectivity can be generally carried out without major structural reconstruction (36). Our results are also in line with studies from medium-chain acyl-CoA dehydrogenase, where it was demonstrated that a functional glutamate involved in substrate dehydrogenation can be relocated in the enzyme active site by a double amino acid substitution (37,38). The results presented here underline the important role of acidic residues for the function of VAO.…”

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

“…Although it is not possible to determine the exact occupancies of the bound ligand in the crystal structures of each complex due to the relatively lower resolution, it is evident, from the levels of electron densities in the final 2F o -F c maps and the refined temperature factors, that the occupancies of the bound ligands in the C12-and C14-CoA complexes are much lower than that of the C8-CoA complex, again implying that the longer product (C12-and C14-CoA) binds less tightly to (or dissociates more easily from) the enzyme. Furthermore, the charge transfer complex formed between MLCADH with 3-thiaoctanoyl-CoA is less stable compared to that between MCADH with the substrate analog (Nandy et al, 1996). Again, the stability of the charge transfer complex is probably directly related to the binding energy of the analog to the active site of the enzyme.…”

“…The "extra space" in the active site cavity is generated by the protein in the case of MLCADH, whereas it is from the lack of the carbonyl oxygen of the bound ligand in the thioether complex. On the other hand, the dramatic increase in the oxygen reactivity with substrate longer than C10-CoA observed in both the wild type and MLCADH can be directly correlated with the product dissociation (Srivastava et al, 1995;Nandy et al, 1996). Although it is not possible to determine the exact occupancies of the bound ligand in the crystal structures of each complex due to the relatively lower resolution, it is evident, from the levels of electron densities in the final 2F o -F c maps and the refined temperature factors, that the occupancies of the bound ligands in the C12-and C14-CoA complexes are much lower than that of the C8-CoA complex, again implying that the longer product (C12-and C14-CoA) binds less tightly to (or dissociates more easily from) the enzyme.…”

“…Another distinction between the action of Glu376 and of Glu255 lies in their interactions with 2-octynoyl-CoA. MCADH is irreversibly inhibited by 2-octynoyl-CoA by covalent modification at Glu376 (Powell & Thorpe, 1988), but neither beef liver LCADH (Ankele et al, 1991) nor MLCADH (Nandy et al, 1996) is covalently modified by the inhibitor. Although 2-octynoyl-CoA is shown to be a mechanism-based inhibitor involving rate-limiting removal of one of the C γ protons (Lau et al, 1988), the exact site of the covalent linkage with the glutamate on the inhibitor is not known at present.…”

“…In the preceding paper, a converse experiment has been performed in which both Glu376 and Thr255 of MCADH have been altered to the corresponding residues found in LCADH, glycine, and glutamate, respectively. The resulting double mutant (Glu376Gly/Thr255Glu) has its substrate specificity and turnover numbers similar to those of LCADH (Nandy et al, 1996, the preceding paper), thus making MCADH specific for medium long chain acyl-CoA substrates (MLCADH). We have determined the three-dimensional structures of MLCADH with bound substrates having various fatty-acyl chain lengths.…”