Search

Plants, lower eukaryotes, bacteria, and archaebacteria synthesise L-histidine (His) in a similar, multistep pathway that is absent in mammals. This makes the His biosynthetic route a promising target for herbicides, antifungal agents, and antibiotics. The last enzyme of the pathway, bifunctional L-histidinol dehydrogenase (HDH, EC 1.1.1.23), catalyses two oxidation reactions: from L-histidinol (HOL) to L-histidinaldehyde and from L-histidinaldehyde to His. Over the course of the reaction, HDH utilises two molecules of NAD + as the hydride acceptor. The object of this study was the HDH enzyme from the model legume plant, Medicago truncatula (MtHDH). Three crystal structures complexed with imidazole, HOL, and His with NAD + provided in-depth insights into the enzyme architecture, its active site, and the cofactor binding mode. The overall structure of MtHDH is similar to the two bacterial orthologues whose three-dimensional structures have been determined. The three snapshots, with the MtHDH enzyme captured in different states, visualise structural rearrangements that allow for NAD + binding for the first time. Furthermore, the MtHDH complex with His and NAD + displays the cofactor molecule situated in a way that would allow for a hydride transfer.

Brucella suis histidinol dehydrogenase (HDH) can be efficiently targeted by substrate analogues. The growth of this pathogen in minimal medium was inhibited and the multiplication in human macrophages was totally abolished in the presence of the drugs. These effects have been shown to be correlated with the previously described inhibition of Brucella HDH activity.

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site., (Copyright 2001 Academic Press.)

Histidinol dehydrogenase (HDH) is one of the enzymes involved in the L-histidine biosynthesis pathway. HDH is a dimer that contains one Zn2+ ion in each identical subunit. In this study, we predicted a possible binding conformation of the intermediate L-histidinal, which is experimentally not known, using a computational modeling method and three potent HDH inhibitors whose structures are similar to that of L-histidinal. At first, a set of the most probable active conformations of the potent inhibitors was determined using two different pharmacophore mapping techniques, the active analogue approach and the distance comparison method. From the most probable active conformations of the three potent inhibitors, the common parts of the L-histidinal structure were extracted and refined by energy minimization to obtain the binding conformation of L-histidinal. This predicted conformation of L-histidinal agrees with an experimentally determined conformation of L-histidine in a single crystal, suggesting that it is an experimentally acceptable conformation. The capability in this conformation to coordinate a Zn2+ ion was examined by comparing the spatial relative geometry of its functional groups with those of ligands that coordinate with a Zn2+ ion in Zn proteins of the Protein Data Bank. This comparison supported our predicted conformation.

Cabbage histidinol dehydrogenase (HDH) oxidizes L-histidinol to L-histidine through two sequential NAD(+)-linked reactions via an alkaline-labile, L-histidinaldehyde intermediate. The kinetic mechanism of the overall reaction as well as the partial reactions involved in the overall catalysis were investigated at pH 7.2 using L-histidinaldehyde as a substrate. Product inhibition patterns conformed to a Bi Uni Uni Bi Ping Pong mechanism as reported for the HDH from Salmonella typhimurium. Thus, the reaction scheme is ordered with the binding of histidinol first and NAD+ second, and histidine is the last product to be released. The intermediate, L-histidinaldehyde, could be a substrate for both the oxidation and the reduction reactions to produce histidine and histidinol, respectively. L-Histidine was not enzymatically reduced in the presence of NADH, indicating that the reaction to oxidize histidinaldehyde is apparently irreversible. L-Histidinaldehyde exhibited a three times greater binding rate constant than histidinol with a considerably small dissociation constant. These results were in agreement with the observation that histidinaldehyde was not released during the overall reaction. The rate of the reduction of histidinaldehyde to histidinol was almost same as that of the overall oxidation reaction. The overall oxidation from histidinol to histidine proceeded about three times slower than the partial oxidation from histidinaldehyde to histidine, suggesting that the first-half forward reaction is the rate-determining step in the total reaction of cabbage HDH.

Histidine ammonia-lyase from Streptomyces griseus was inactivated by methylglyoxal and phenylglyoxal, dicarbonyl reagents known to react specifically with arginyl residues in proteins. The inactivation showed pseudo-first-order kinetics and could be prevented by protection with histidinol phosphate, a competitive inhibitor of histidine ammonia-lyase. Analysis of the amino acid composition of histidine ammonia-lyase after treatment with phenylglyoxal, together with the kinetics of inactivation, suggested that inactivation was a consequence of specific reaction with one or more essential arginyl residues at or near the active site of the enzyme.

Histidine ammonia-lyase (histidase) was purified to homogeneity from vegetative mycelia of Streptomyces griseus. The enzyme was specific for L-histidine and showed no activity against the substrate analog, D-histidine. Histidinol phosphate was a potent competitive inhibitor. Histidase displayed saturation kinetics with no detectable sigmoidal response. Neither thiol reagents nor a variety of divalent cations had any effect on the activity of the purified enzyme. High concentrations of potassium cyanide inactivated histidase in the absence of its substrate or histidinol phosphate, suggesting that, as in other histidases, dehydroalanine plays an important role in catalysis. The N-terminal amino acid sequence of histidase was used to construct a mixed oligonucleotide probe to identify and clone the histidase structural gene, hutH, from genomic DNA of the wild-type strain of S. griseus. The cloned DNA restored the ability of a histidase structural gene mutant to grow on L-histidine as the sole nitrogen source. The deduced amino acid sequence of hutH shows significant relatedness with histidase from bacteria and a mammal as well as phenylalanine ammonia-lyase from plants and fungi.

One molecule of the enzymatic intermediate histidinal is firmly bound per subunit of histidinol dehydrogenase (EC 1.1.1.23) and protected against decomposition. The dissociation rate constant of the histidinal--histidinol dehydrogenase complex is estimated as 2.5 X 10(-5) S-1. Steady-state kinetic measurements studying the oxidation of histidinal to histidine and the reduction of histidinal to histidinol allow to calculate the association rate constants for histidinal. For both reactions the association rate constant is found as 1.9 X 10(6) M-1 S-1. Thus the dissociation constant of the histidinal--histidinol dehydrogenase complex is estimated to be of the order of 1.4 X 10(-11) M.