The Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated byN-Propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction,

Oxidation of amino acids, like proline catabolism, is a central part of energy metabolism. Proline is oxidized to glutamate by two enzymes: proline dehydrogenase (PRODH) and 1-pyrroline-5-carboxylate dehydrogenase (P5CDH). PRODH catalyzes the first reaction of proline to 1-pyrroline-5-carboxylate (P5C). P5C undergoes a non-enzymatic hydrolysis to glutamate semialdehyde (GSA), which is oxidized to glutamate by a NAD+- dependent enzyme P5CDH. PRODH and P5CDH are mono-functional enzymes in eukaryotes and Gram-positive bacteria; while in Gram-negative bacteria, the two enzymes are fused into one protein as two domains, known as proline utilization A (PutA). This dissertation work involved structural and functional studies of PRODH, P5CDH, PutA, and human aldehyde dehydrogenases (ALDHs). The results illuminated the substrate recognition for mono-functional PRODH and hot spot oligomerization mechanism for mono-functional P5CDH, also, demonstrated that diethylaminobenzaldehyde (DEAB) is a mechanism based inactivator for aldehyde dehydrogenase 7A1. Furthermore, the C-terminal domain found in PutAs, the only domain without any structural and functional information has been structurally and biochemically characterized.

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Involvement of the β3-α3 Loop of the Proline Dehydrogenase Domain in Allosteric Regulation of Membrane Association of Proline Utilization A

Biochemistry ◽

10.1021/bi400396g ◽

2013 ◽

Vol 52 (26) ◽

pp. 4482-4491 ◽

Cited By ~ 14

Author(s):

Weidong Zhu ◽

Ashley M. Haile ◽

Ranjan K. Singh ◽

John D. Larson ◽

Danielle Smithen ◽

...

Keyword(s):

Allosteric Regulation ◽

Membrane Association ◽

Proline Dehydrogenase ◽

Proline Utilization ◽

Proline Utilization A

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Copper–oxygen Dynamics in Tyrosinase

10.26434/chemrxiv.9255251 ◽

2019 ◽

Author(s):

Nobutaka Fujieda ◽

Sachiko Yanagisawa ◽

Minoru Kubo ◽

Genji Kurisu ◽

Shinobu Itoh

Keyword(s):

Catalytic Mechanism ◽

Substrate Binding ◽

Active Form ◽

Copper Ion ◽

Atomic Resolution ◽

Oxygen Species ◽

X Ray ◽

Oxygen Dynamics ◽

Insight Into ◽

Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu2O2core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.

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Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4

Glycobiology ◽

10.1093/glycob/cwab025 ◽

2021 ◽

Author(s):

Margrethe Gaardløs ◽

Sergey A Samsonov ◽

Marit Sletmoen ◽

Maya Hjørnevik ◽

Gerd Inger Sætrom ◽

...

Keyword(s):

Optical Tweezers ◽

Conformational Changes ◽

Molecular Mechanisms ◽

Hydrophobic Interactions ◽

Substrate Binding ◽

Interdisciplinary Approach ◽

Product Formation ◽

Titration Calorimetry ◽

Binding Groove ◽

Dynamics Simulations

Abstract Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

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