Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/μ-Crystallin Under Physiological Conditions

Abstract

Mammalian ketimine reductase is identical to μ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity

“…Arabidopsis At5g52810 (SARD4, alias ORNCD1) represents the Arabidopsis gene with the highest sequence similarity to CRYM (30.5% amino acid identity and 51.8% amino acid similarity; EMBOSS NeedlemanWunsch pairwise alignment, version 6.3.1, EMBL-EBI; http://www.ebi.ac.uk/Tools/psa/; Zeier, 2013), the ketimine reductase that is able to reduce 1,2-DP to Pip (Hallen et al, 2011(Hallen et al, , 2015. Similar to CRYM, SARD4 possesses a putative NAD(P)-binding domain with a predicted Rossmann fold, a structural motif frequently found in nucleotide-binding dehydrogenases (Rao and Rossmann, 1973;Zeier, 2013).…”

“…S11). Mammalian CRYM possesses a similar Pyr2C reductase activity (Hallen et al, 2015;Supplemental Fig. S11).…”

“…SARD4 is the closest homolog of the mammalian ketimine reductase CRYM that utilizes either NADH or NADPH as a cofactor (Hallen et al, 2011). CRYM preferentially catalyzes the reduction of cyclic ketimine monocarboxylate substrates such as 1,2-DP or its five-membered ring analog D 1 -Pyr2C (Hallen et al, 2015). Subcellular localization experiments indicate that, like ALD1, Arabidopsis SARD4 resides in plastids (Sharma et al, 2013).…”

“…Since CRYM has been characterized as a reductase that accepts ketiminic substrates such as 1,2-DP (Hallen et al, 2015), we assume that the 2,3-DP detected in our assays and plant extracts is not the direct substrate for SARD4 (and CRYM). Instead, an equilibrium between 2,3-DP and 1,2-DP that is largely shifted to the detectable enaminic form could exist.…”

“…Later, partially purified enzyme preparations from different mammalian tissues with imine reductase activities toward several cyclic ketimines and the ability to convert 1,2-DP to Pip were obtained Nardini et al, 1988;Hallen et al, 2013). More recently, the mammalian protein m-crystallin (CRYM) was characterized as an NAD(P)H-dependent ketimine reductase that is able to reduce 1,2-DP to the supposed product L-Pip (Hallen et al, 2011(Hallen et al, , 2015.…”