Abstract
Thiolases are a well characterized family of enzymes with two distinct categories: degradative, β-ketoadipyl-CoA thiolases and biosynthetic, acetoacetyl-CoA thiolases. Both classes share an identical catalytic triad but catalyze reactions in opposite directions. Moreover, it is established that in contrast to the biosynthetic thiolases the degradative thiolases can accept substrates with broad chain lengths. Hitherto, no residue or structural pattern has been recognized that might help to discern the two thiolases, here we exploit, a tetrameric degradative thiolase from Pseudomonas putida KT2440 annotated as PcaF, as a model system to understand features which distinguishes the two classes using structural studies and bioinformatics analyses. Degradative thiolases have different active site architecture when compared to biosynthetic thiolases, demonstrating the dissimilar chemical nature of the active site architecture. Both thiolases deploy different “anchoring residues” to tether the large Coenzyme A (CoA) or CoA derivatives. Interestingly, the H356 of the catalytic triad in PcaF is directly involved in tethering the CoA/CoA derivatives into the active site and we were able to trap a gridlocked thiolase structure of the H356A mutant, where the CoA was found to be covalently linked to the catalytic cysteine residue, inhibiting the overall reaction. Further, X-ray structures with two long chain CoA derivatives, hexanal-CoA and octanal-CoA helped in delineating the long tunnel of 235 Å2 surface area in PcaF and led to identification of a unique covering loop exclusive to degradative thiolases that plays an active role in determining the tunnel length and the nature of the binding substrate.
Highlights
Thiolases, known as acetyl-coenzyme A acetyltransferases, are a prevalent class of enzymes that are found in both prokaryotes and eukaryotes
The 1.81 Å apo β-ketoadipyl-Coenzyme A (CoA) thiolase (PcaF) structure was solved by the molecular replacement (MR) method using a putative acetyl-CoA acetyltransferase (PDB code: 1ULQ, 52% sequence identity)
In the Zr-thiolases, H157 acts as one of the anchoring residues and is conserved across the biosynthetic thiolases, but in the case of degradative thiolases, the residue at this position varies, implying that this position does not participate in substrate selection
Summary
Known as acetyl-coenzyme A acetyltransferases, are a prevalent class of enzymes that are found in both prokaryotes and eukaryotes. Degradative or 3-ketoacyl-CoA thiolases are involved in the thiolytic breakdown of β-ketoacyl-CoA to acetyl-CoA and shorter acyl CoA molecule (Haapalainen et al, 2006; Mathieu et al, 1997) and can accommodate long acyl chain substrates. This catalytic cleavage by degradative thiolases is vital as it accounts for the energy production in cells.
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