Cloning, Expression, and Characterization of a Human 4′-Phosphopantetheinyl Transferase with Broad Substrate Specificity

Vangipuram S Rangan,Stuart Smith,Lei Zhang,Anil K Joshi

doi:10.1074/jbc.m305459200

Abstract

A single candidate 4'-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4'-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4'-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the alpha-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336-342). Thus, in contrast to yeast, which utilizes separate 4'-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4'-phosphopantetheinylation reactions.

Highlights

The fatty acid synthases (FASs)1 associated with the soluble cytoplasm of yeast and animal cells comprise large multifunctional polypeptides that contain all of the catalytic components required for the synthesis of long-chain fatty acids from malonyl-CoA de novo
Expression, and Purification of the Human Mitochondrial acyl carrier protein (ACP) (ACPmit)—Based on BLAST searches of the human genome sequence data base, using authentic type I and type II ACPs, we identified a putative ACPmit sequence that was represented in two human EST clones (IMAGE clones 4799845 and 4816243)
BLAST searching with a phosphopantetheine transferase (PPTase) probe sequence of the high molecular weight type, namely the B. subtilis surfactin synthetase-activating enzyme, Sfp, revealed a candidate human PPTase sequence that was subsequently used to derive full-length cDNAs, by PCR, from human liver and brain cDNA libraries and from a human EST cDNA clone

Summary

Introduction

The fatty acid synthases (FASs) associated with the soluble cytoplasm of yeast and animal cells comprise large multifunctional polypeptides that contain all of the catalytic components required for the synthesis of long-chain fatty acids from malonyl-CoA de novo. These multifunctional polypeptides are commonly referred to as type I FASs. The animal FASs consist of two identical polypeptides of approximately 2500 residues (␣2), whereas the yeast FAS comprises six copies each of two non-. The former is a constituent domain of the ␣-subunit of the type I cytosolic FAS, so that this protein is capable of selfphosphopantetheinylation [16], whereas the latter is a separate PPTase that acts only on the mitochondrial ACP [17]

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