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Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active center base arrangement
A Nandy1, V Kieweg, F G Kräutle
1Faculty of Biology, University of Konstanz, Germany.
Insights
Researchers engineered a chimeric enzyme, medium-/long-chain acyl-CoA dehydrogenase (MLCADH), by altering key glutamate residues in medium-chain acyl-CoA dehydrogenase (MCADH). This modified enzyme shows altered substrate specificity and reactivity, offering insights into acyl-CoA dehydrogenase function.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Protein Structure-Function Relationships
Background:
- Acyl-CoA dehydrogenases (ACADs) are crucial enzymes in fatty acid metabolism.
- The catalytic glutamate residue's position influences substrate specificity and reactivity.
- Medium-chain acyl-CoA dehydrogenase (MCADH), long-chain acyl-CoA dehydrogenase (LCADH), and isovaleryl-CoA dehydrogenase (IVDH) have distinct active site topologies.
Purpose of the Study:
- To investigate the role of catalytic glutamate residue topology in ACAD function.
- To create a chimeric enzyme (MLCADH) by transferring LCADH topology to MCADH.
- To analyze the impact of these modifications on enzyme activity, substrate specificity, and reactivity.
Main Methods:
- Site-directed mutagenesis was used to introduce two mutations (Glu376Gly and Thr255Glu) into MCADH, creating the chimeric MLCADH.
- Enzyme activity assays were performed using various acyl-CoA substrates (octanoyl-CoA, dodecanoyl-CoA).
- Reoxidation rates with oxygen were measured to assess enzyme kinetics.
Main Results:
- The chimeric MLCADH exhibited approximately 20% of MCADH activity and 25% of LCADH activity with their respective optimal substrates.
- MLCADH showed an enhanced rate of reoxidation with oxygen.
- A significantly narrower substrate chain length specificity was observed for MLCADH, peaking with dodecanoyl-CoA, similar to LCADH.
Conclusions:
- The topology of the catalytic glutamate residue is a critical determinant of substrate chain length specificity and reactivity in ACADs.
- Engineering enzyme topology can modulate substrate specificity and kinetic properties.
- The findings provide insights into the evolutionary relationships and functional diversification of ACADs.
Abstract:
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].