Full-length rat HDC (L-histidine decarboxylase) translated in reticulocyte cell lysate reactions is inactive, whereas C-terminally truncated isoforms are capable of histamine biosynthesis. C-terminal processing of the approximately 74 kDa full-length protein occurs naturally in vivo, with the production of multiple truncated isoforms. The minimal C-terminal truncation required for the acquisition of catalytic competence has yet to be defined, however, and it remains unclear as to why truncation is needed. Here we show that approximately 74 kDa HDC monomers can form dimers, which is the conformation in which the enzyme is thought to be catalytically active. Nevertheless, the resulting dimer is unable to establish pyridoxal phosphate-dependent interactions with an L-histidine substrate analogue. Protein sequences localized to between amino acids 617 and 633 specifically mediate this inhibition. Removing this region or replacing the entire C-terminus with non-HDC protein sequences permitted interactions with the substrate analogue to be re-established. This corresponded exactly with the acquisition of catalytic competence, and the ability to decarboxylate natural L-histidine substrate. These studies suggested that the approximately 74 kDa full-length isoform is deficient in substrate binding, and demonstrated that C-terminally truncated isoforms with molecular masses between approximately 70 kDa and approximately 58 kDa have gradually increasing specific activities. The physiological relevance of our results is discussed in the context of differential expression of HDC isoforms in vivo.

HDC (L-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from L-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme L-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an L-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.