CRAT

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Introduction

Carnitine acyltransferases are responsible for catalyzing the acyl groups exchange between carnitine and coenzyme A (CoA). These enzymes include carnitine acetyltransferase (CRAT, also known as CAT), carnitine palmitoyltransferase (CPT) and carnitine octanoyltransferase (COT), which have preferences for substrates of short-chain, medium-chain, and long-chain fatty acids. These enzymes usually contain about 600 residues with molecular weights of about 70 kDa. Carnitine acyltransferases have important biological functions. For example, CPT-I and CPT-II promote the transport of long-chain fatty acids across the mitochondrial membrane, and therefore play a vital role in the β-oxidation of long-chain fatty acids in the mitochondria. CRAT may be involved in the transport of acetyl-CoA across the cell membrane to maintain the balance of acetyl-CoA: CoA. In addition, CRAT also participates in the progression through G1 to the S phase of the cell cycle.

Mutations or disorders of these enzymes are related to many serious and even fatal human diseases. For example, the Inherited recessive defects of CPT-I and CPT-II can cause hypoketonemia and hypoglycemia, and CPT-II deficiency is the most common cause of abnormal lipid metabolism in skeletal muscle. Inherited defects in CRAT activity can lead to serious heart and neurological problems. In patients with Alzheimer's disease, CRAT exhibits decreased activity. Therefore, carnitine acyltransferases have become promising targets for the development of therapeutic agents against several human diseases. For example, inhibitors of L-CPT-I are isotypes of CPT-I, which is mainly expressed in the liver and kidney, and can effectively treat non-insulin-dependent diabetes mellitus (NIDDM). Unfortunately, the clinical application of these irreversible inhibitors is severely limited by their side effects. There is no recognizable sequence homology between these enzymes and other proteins in the database, leading to a challenging process for the design and development of new and reversible inhibitors of L-CPT-I, so a better understanding of the structure and function of these enzymes is needed.

Overall Structure of CRAT

The crystal structure of free enzyme of mouse CRAT at 1.8 Å resolution has been obtained. The atomic model contains 30-625 for each of the two molecules in the asymmetric unit, which basically corresponds to the full-length mature form of the enzyme. All the residues except IIe116 are located in the favored regions of the Ramachandran plot. CRAT consists of 16 β chains (β1-β16) and 20 α helices (α1-α20), which can be divided into two domains. The C domain contains a six-stranded mixed β sheet and eleven α helices. The residues in this domain include residues 407-626 at the C-terminus and residues 30-95 at the N-terminus of the protein and one face of the β sheet in this domain is covered by the α helices. Helix α13, the region of residues 386-487, forms a long connection between the N and C domains.

The N domain contains an eight-strand mixed β sheet, which is covered by eight α helices on both sides. Surprisingly, the N and C domains share very similar polypeptide backbone folds, despite the lack of any recognizable amino acid sequence homology between them. This structural homology is limited to the core of the two domains, including the six β strands in the center of the β sheet and the three closely related α helices (α6, α7, and α12). A total of 71 Cα positions can be superimposed to within 3.5 Å between the two domains, and the root mean square distance for these equivalent atoms is 2.0 Å.

Figure 1. Structure of CRAT (Gerwald, J.; Liang, T. 2003)

Implications for the Catalytic Mechanism: Substrate-Assisted Catalysis

The His residue in the active site of CRAT serves as a general base in the catalysis. Depending on the direction of the reaction, it extracts protons from the 3-hydroxyl group of carnitine or the thiol group of CoA. It can be clearly seen from the mechanism and structure that when only acylcarnitine or acyl-CoA binds to the active site, a water molecule will bind in the opposite channel of the active site and become the receptor for the acyl group. Therefore, these transferases can also catalyze the simple hydrolysis of acylcarnitine or acyl-CoA. This futile hydrolysis may not be a significant reaction in vivo because the active site of the enzyme may be occupied by its two substrates. Structural analysis and modeling studies indicate that the trimethylammonium group of carnitine may play an important role in the catalysis by these acyltransferases by stabilizing the oxyanion in the tetrahedral intermediate of the reaction. This is supported by the observation that the positive charge of carnitine is not important for its binding, but it is necessary for the catalytic function.

Figure 2. The Catalytic Mechanism of Carnitine Acyltransferases (Gerwald, J.; Liang, T. 2003)