Functional Versatility of the Human 2-Oxoadipate Dehydrogenase in the L-Lysine Degradation Pathway toward Its Non-Cognate Substrate 2-Oxopimelic Acid
Abstract
:1. Introduction
2. Results and Discussion
2.1. The E1a Oxidizes 2-Oxopimelic Acid at Rates Similar to the Rates with Its Substrate 2-Oxoadipate through Decarboxylation and Enamine Intermediate Formation
(E1a-Thdp-enamine intermediate) + CO2
S8-glutaryldihydrolipoyl-E2o (reductive glutarylation)
2.2. The E1a-ThDP-Enamine Intermediate Formed on Decarboxylation of OP Is Catalytically Competent in the E1a-Side Reactions and Could Be an Efficient Source of H2O2 Production
2.3. Generation of NADH from OP Is Strongly Diminished Due to the Diminished Rate of Reductive Transfer of the Adipoyl Moiety from the E1a to the E2o Component Affected by One-Carbon Substrate Elongation
2.4. Binding Models of E1a with OP and of E2o with S8-Adipoyldihydrolipoamide in the Active Centers
2.5. The E1a-Specific Activity and the E1a Side-Reactions with OP Are Affected by E1a Substitutions Associated with AMOXAD and EoE and Showed a Great Similarity to the Behavior with OA
2.5.1. Functional Characterization of the E1a Variants with Disease-Causing Mutations
2.5.2. Studies on E1a Variants Encoded by DHTKD1 with Genetic Mutations Linked to AMOXAD and EoE
2.5.3. Evidence for the Formation of a Pre-Decarboxylation Intermediate from OP by E1a Variants with Mutations
3. Materials and Methods
3.1. Protein Expression and Purification
3.2. Overall Activity of NADH Production upon Assembly of E1a with E2o and E3
3.3. E1a-Specific Activity
3.4. Measurement for Formation of Adipoyl Semialdehyde by E1a from OP
3.5. Measurement of the Rate of H2O2 Production by Fluorescence Spectroscopy
3.6. Circular Dichroism Spectroscopy for Detection of Carboligase Products, (S)-2-Hydroxy-3-Oxo-Octanedioic and (S)-8-Ethoxy-7-Hydroxy-6,8-Dioxo-Octanoic Acid
3.7. Circular Dichroism Titration of E1a and E1a Variants by Adipoylphosphonic Acid and by Thiamin 2-Thiothiazolone Diphosphate
3.8. Reductive Acylation of LDo by E1a and OA or OP In Vitro as Detected by Fourier Transform Mass Spectrometry
3.9. Enzymatic Synthesis of Adipoyl-CoA
3.10. Fluorescence Titration of Pyrene-Labeled E2o1–173 Di-Domain by Arg715Cys E1a
3.11. Ligand Docking
4. Conclusions
- The functional promiscuity of the human E1a component has been demonstrated and adds to our understanding of the OADHc function in the L-lysine catabolism pathway. According to biochemical and structural findings, the E1a active centers accept OP with a one-carbon longer chain compared to the natural substrate OA, with no significant effect on the rate of decarboxylation and on the formation of the E1a-ThDP-enamine intermediate, as well as on all three side-reactions derived from the E1a-enamine intermediate. Importantly, the findings indicate that E1a is a better source of H2O2 production from OA and OP than E1o with its natural substrate OG, a finding that may potentially affect mitochondrial structure and function.
- The E2o component, an important enzyme in the TCA cycle, is known as a gatekeeper in intermediate channeling in 2-oxo acid dehydrogenase complexes [29,30,46,47,48,49,50]. Our findings suggest involvement of the E2o in the rate-limiting step at two levels in the mechanism, specifically, by controlling the rate of transfer of the acyl group between the E1a and E2o components (so called reductive acylation) as generally accepted, and by controlling the rate of transfer of the acyl group from S8-acyldihydrolipoyl-E2o to CoA in its active centers. At the same time, the rate of recycling of dihydrolipoyl-E2o to lipoyl-E2o proceeds faster [26,40,41].
- Evidence is presented that the E2o component of the 2-oxoglutarate dehydrogenase complex in the TCA cycle can produce acyl-CoA thioesters of different lengths, such as succinyl-CoA (C4), glutaryl-CoA (C5) and, less efficiently, adipoyl-CoA (C6), indicating its promiscuity, not recognized before.
- The E1a variants with mutations associated with AMOXAD and EoE affected the E1a-specific and the E1a-enamine side reactions with OP and showed a great similarity to that observed with OA. The mechanism underlying diminished E1a activity due to the Arg715Cys substitution is suggested. The data point to local conformational changes in the C-terminal region of the E1a homodimer, with consequences for ThDP cofactor and substrate binding, as well as for its assembly with E2o and E3.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Overall NADH Activity a,e | E1a-Specific Activity b | ||
---|---|---|---|
μmol·min−1·mg E1a−1 | μmol·min−1·mg E1a−1 | ||
OA | OP | OA | OP |
1.38 ± 0.25 | 0.012 ± 0.002 | 1.86 ± 0.09 | 1.56 ± 0.15 |
kcat = 4.8 ± 0.9 s−1 | kcat = 0.04 ± 0.003 s−1 | kcat = 6.5 s−1 | kcat = 5.4 s−1 |
kcat/Km = 400 × 103 M−1 s−1 | kcat/Km = 1.1 × 103 M−1 s−1 | kcat/Km = 1326 × 103 M−1 s−1 | kcat/Km = 146 × 103 M−1 s−1 |
Glutarylation/Adipoylation c | Glutaryl-CoA formation d | Adipoyl-CoA formation d | |
kglutaryl = 8.2 s−1 | kadipoyl= 0.028 s−1 | kglutaryl-CoA = 0.245 μM−1 s−1 | kadipoyl-CoA = 0.003 μM−1 s−1 |
E1a carboligation activity f | H2O2 activity of E1a f | ||
μmol ·min−1·mg E1a−1 | nmol min−1∙mg E1a−1 | ||
OA | OP | OA | OP |
1.04 ± 0.17 | 1.46 ± 0.05 | 8.44 ± 1.69 | 8.36 ± 0.78 |
kcat = 3.6 ± 0.41 s−1 | kcat = 5.1 ± 0.15 s−1 | kcat = 0.029 ± 0.006 s−1 | kcat = 0.029 ± 0.003 s−1 |
kcat/Km = 736 × 103 M−1 s−1 | kcat/Km = 138 × 103 M−1 s−1 | kcat/Km = 5.9 × 103 M−1 s−1 | kcat/Km = 0.78 × 103 M−1 s−1 |
Glutaric acid semialdehyde formation f | Adipic acid semialdehyde formation f | ||
nmol·min−1·mg E1a−1 | nmol·min−1·mg E1a−1 | ||
6.05 ± 0.46 | 5.7 ± 1.1 | ||
kcat = 0.021 ± 0.002 s−1 | kcat = 0.020 ± 0.003 s−1 |
E1a Substitutions | E1a-Specific Activity c (μmol min−1·mg E1a−1) | H2O2 Activity (nmol·min−1·mg E1a−1) | E1a Carboligation Activity (μmol·min−1·mg E1a−1) | |||
---|---|---|---|---|---|---|
OA | OP | OA | OP | OA | OP | |
None a | 1.86 ± 0.09 (100%) | 1.56 ± 0.15 (100%) | 8.44 ± 1.69 (100%) | 8.37 ± 0.78 (100%) | 1.04 ± 0.17 (100%) | 1.46 ± 0.05 (100%) |
E1a variants associated with AMOXAD | ||||||
Leu234Gly | 0.081 ± 0.007 (4.35%) | 0.091 ± 0.01 (5.8%) | 0.085 ± 0.024 (1.0%) | 0.070 ± 0.002 (0.84%) | 0.038 ± 0.006 (3.7%) | 0.041 ± 0.009 (2.8%) |
Gln305His | 1.23 ± 0.03 (66%) | 1.00 ± 0.04 (64%) | 5.42 ± 0.89 (64%) | 8.37 ± 0.47 (100%) | 1.01 ± 0.11 (97%) | 1.34 ± 0.03 (92%) |
Arg455Gln | 0.62 ± 0.09 (33%) | 0.352 ± 0.019 (23%) | 0.054 ± 0.004 (0.6%) | 0.32 ± 0.07 (3.8%) | 0.048 ± 0.012 (4.6%) | 0.043 ± 0.001 (3%) |
Arg715Cys | 0.009 ± 0.001 (0.5%) | 0.007 ± 0.001 (0.45%) | 0.034 ± 0.002 (0.4%) | N/D | 0.02 ± 0.003 (1.9%) | 0.014 ± 0.002 (0.96%) |
Pro773 Leu | 0.592 ± 0.031 (32%) | 0.543 ± 0.037 (35%) | 3.27 ± 0.11 (39%) | 4.88 ± 0.13 (58%) | 0.70 ± 0.02 (67%) | 0.902 ± 0.047 (62%) |
E1a variants associated with EoE | ||||||
None b | 0.54 ± 0.026 (100%) | 0.47 ± 0.01 (100%) | 1.41 ± 0.10 (100%) | 1.69 ± 0.08 (100%) | 0.268 ± 0.027 (100%) | 0.214 ± 0.013 (100%) |
Arg163Gln | 0.037 ± 0.001 (6.9%) | 0.020 ± 0.004 (4.3%) | 0.113 ± 0.003 (8%) | 0.111 ± 0.004 (6.5%) | 0.021 ± 0.005 (7.8 %) | 0.012 ± 0.003 (5.6%) |
Val360Ala | 0.195 ± 0.030 (36%) | 0.142 ± 0.007 (30%) | 0.29 ± 0.02 (21%) | 0.35 ± 0.030 (21%) | 0.085 ± 0.006 (32%) | 0.070 ± 0.02 (32.7%) |
Ser862Ile | 0.571 ± 0.023 (100%) | 0.48 ± 0.01 (100%) | 1.08 ± 0.09 (77%) | 1.28 ± 0.05 (76%) | 0.296 ± 0.008 (100%) | 0.202 ± 0.029 (94%) |
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Nemeria, N.S.; Nagy, B.; Sanchez, R.; Zhang, X.; Leandro, J.; Ambrus, A.; Houten, S.M.; Jordan, F. Functional Versatility of the Human 2-Oxoadipate Dehydrogenase in the L-Lysine Degradation Pathway toward Its Non-Cognate Substrate 2-Oxopimelic Acid. Int. J. Mol. Sci. 2022, 23, 8213. https://doi.org/10.3390/ijms23158213
Nemeria NS, Nagy B, Sanchez R, Zhang X, Leandro J, Ambrus A, Houten SM, Jordan F. Functional Versatility of the Human 2-Oxoadipate Dehydrogenase in the L-Lysine Degradation Pathway toward Its Non-Cognate Substrate 2-Oxopimelic Acid. International Journal of Molecular Sciences. 2022; 23(15):8213. https://doi.org/10.3390/ijms23158213
Chicago/Turabian StyleNemeria, Natalia S., Balint Nagy, Roberto Sanchez, Xu Zhang, João Leandro, Attila Ambrus, Sander M. Houten, and Frank Jordan. 2022. "Functional Versatility of the Human 2-Oxoadipate Dehydrogenase in the L-Lysine Degradation Pathway toward Its Non-Cognate Substrate 2-Oxopimelic Acid" International Journal of Molecular Sciences 23, no. 15: 8213. https://doi.org/10.3390/ijms23158213