X-ray crystallographic studies of enzymes by molecular replacement

Abstract

The structures of four enzymes, namely human ubiquitous R167Q porphobilinogen deaminase (uPBGD). bovine inositol monophosphatase (IMPase), D303E methanol dehydrogenase (MDH) from 2-hydroxy-6-keto-nona-2,4-diene-l,9-dioic acid 5,6-hydrolase Methylobacterimn extorqiiens (MhpC) from E. coli have been solved by X-ray crystallography using molecular replacement. and P B GD is the third enzyme in the haem biosynthetic pathway and assembles four molecules of porphobilinogen to form the linear tertrapyrrole preuroporphyrinogen III. The R167Q mutation in human P B GD retains 6-7 % activit\ of the wild type enzyme and is one of those responsible for the hereditary disorder, Initial phases for human R167Q-uPBGD were obtained using the E. coli P B GD as the search model and the structure has been refined to 2.65 A. Preliminar}' structural the dipyrromethane cofactor is in the reduced (active) form covalently linked to Cys-261 in domain 3. A sequence insertion in domain 3 is located after strand (333. It stabilises the domain 1 interface and the hydrophobic core in domain 3. The additional electron density present at the active site cleft may arise from a bound substrate molecule. analysis demonstrates that acute intermittent porphyria. monophosphate to liberate inositol. IMPase is a key enzyme in the phosphatidyl inositol signalling pathway and catalyses the hydrolysis of inositol The enzyme is the putative target for the mood stabilising drug, lithium. A bovine IMPase cry stal grown under inhibitory conditions (20 mM MgCli) with a dimer in the asymmetric unit Initial phases were obtained by molecular replacement with the human IMPase structure as the search model. Although the refined structure shows that each subunit possesses 3 bound Mg"' ions, only two are implicated in the mechanism. The third Mg"^ may diminish the ability of Glu-70 to activate a water molecule. diffracted X-rays to 1.65 A resolution. although the apparent MDH is a periplasmic quinoprotein in methylotrophic bacteria that utilises pyrrolo-quinoline quinone (PQQ) to oxidize methanol to formaldehyde. The reaction is presumably initiated by the abstraction of a proton from methanol by Asp-303. Site directed mutagenesis of the putative nucleophile (D303E) yields an active mutant The D303E-MDH crystal with two tetramers in the asymmetric unit diffracted X-rays to 3.0 A resolution. Initial phases for D303E-MDH were obtained using the wild type structure as the search model. The refined structure of D303E-MDH reveals that the extended Glu-303 side-chain forms new interactions with the C-5 carbonyl group of PQQ and the Ca"' ion, thus reducing its likelihood of abstracting a proton from methanol. The reduced affinity for methanol can be attributed to either steric hindrance by Glu- 303 or to the change in the co-ordination sphere of the Ca"^ ion. K,„ has increased 80-000-fold. MhpC, a bacterial C-C hydrolase involved in the degradation of 3-hydroxy phenylpropionic acid is of great interest to the bioremediation industry. MhpC catalyses the hydrolytic cleavage of a C-C bond of 2-hydroxy-6-keto-nona-2,4-diene-l,9 dioic acid to yield 2-hydroxypenta-2,4-dienoic acid and succinic acid. Attempts to obtain phases by molecular replacement using a number of weak structural homologues proved unsuccessful. A retrospective analysis of MhpC using the co-ordinates from an independent multiple anomalous dispersion analysis suggests that a combination of factors colluded to prevent a successful molecular replacement solution, the most significant being a large structural difference after strand 6 (a series of helices and large connecting loops) between MhpC and the search models employed.

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