Jennifer A. Runquist

Crystal Structure of Human 3-Hydroxy-3-methylglutaryl-CoA Lyase INSIGHTS INTO CATALYSIS AND THE MOLECULAR BASIS FOR HYDROXYMETHYLGLUTARIC ACIDURIA

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme. We report the experimental determination of a crystal structure at 2.1 Å resolution of the recombinant human mitochondrial HMG-CoA lyase containing a bound activator cation and the dicarboxylic acid 3-hydroxyglutarate. The enzyme adopts a (βα)8 barrel fold, and the N-terminal barrel end is occluded. The structure of a physiologically relevant dimer suggests that substrate access to the active site involves binding across the cavity located at the C-terminal end of the barrel. An alternative hypothesis that involves substrate insertion through a pore proposed to extend through the barrel is not compatible with the observed structure. The activator cation ligands included Asn275, Asp42,His233, and His235; the latter three residues had been implicated previously as contributing to metal binding or enzyme activity. Arg41, previously shown to have a major effect on catalytic efficiency, is also located at the active site. In the observed structure, this residue interacts with a carboxyl group of 3-hydroxyglutarate, the hydrolysis product of the competitive inhibitor 3-hydroxyglutaryl-CoA required for crystallization of human enzyme. The structure provides a rationale for the decrease in enzyme activity due to clinical mutations, including H233R, R41Q, D42H, and D204N, that compromise active site function or enzyme stability.

Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 Å, respectively. Comparison of these β/α-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg2+ coordination and positioning of the flexible loop containing the conserved HMGCL “signature” sequence. In the R41M-Mg2+-substrate ternary complex, loop residue Cys266 (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg2+-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg2+ liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His233 and His235 imidazoles, other Mg2+ ligands are the Asp42 carboxyl oxygen and an ordered water molecule. This water, positioned between Asp42 and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg41 with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg41 mutation on reaction product enolization and explains why human Arg41 mutations cause drastic enzyme deficiency.

Functional contribution of a conserved, mobile loop histidine of phosphoribulokinase

In the Rhodobacter sphaeroides phosphoribulokinase (PRK) structure, there are several disordered regions, including a loop containing invariant residues Y98 and H100. The functional importance of these residues has been unclear. PRK is inactivated by diethyl pyrocarbonate (DEPC) and protected by the substrates ATP and Ru5P, as well as by the competitive inhibitor, 6‐phosphogluconate, suggesting active site histidine residue(s). PRK contains only three invariant histidines: H45, H100, and H134. Previous mutagenesis studies discount significant function for H134, but implicate H45 in Ru5P binding. PRK mutant H45N is inactivated by DEPC, implicating a second active site histidine. To evaluate the function of H100, as well as another invariant loop residue Y98, PRK mutants Y98L, H100A, H100N, and H100Q were characterized. Mutant PRK binding stoichiometries for the fluorescent alternative substrate, trinitrophenyl‐ATP, as well as the allosteric activator, NADH, are comparable to wild‐type PRK values, suggesting intact effector and substrate binding sites. The KmRu5P for the H100 mutants shows modest eight‐ to 14‐fold inflation effects, whereas Y98L exhibits a 40‐fold inflation for KmRu5P. However, Y98Ls Ki for the competitive inhibitor 6‐phosphogluconate is close to that of wild‐type PRK. These observations suggest that Y98 and H100 are not essential Ru5P binding determinants. The Vm of Y98L is diminished 27‐fold compared with wild‐type PRK. In contrast, H100A, H100N, and H100Q exhibit significant decreases in Vm of 2600‐, 2300‐, and 735‐fold, respectively. Results suggest that the mobile region containing Y98 and H100 must contribute to PRKs active site. Moreover, H100s imidazole significantly influences catalytic efficiency.

Anionic substitutes for catalytic aspartic acids in phosphoribulokinase

Mutagenic substitution of the invariant D42 and D169 residues in phosphoribulokinase (PRK) with amino acids that contain neutral side chains (e.g., alanine or asparagine) results in large decreases in catalytic efficiency (10(5)- and 10(4)-fold for replacement of D42 and D169, respectively). To further evaluate the importance of anionic side chains at residues 42 and 169, substitutions of glutamic acid (D42E, D169E) and cysteine (D42C and D169C in an otherwise cysteine-free protein) have been engineered. All purified mutant enzymes bind the fluorescent alternative substrate trinitrophenyl-ATP and the allosteric effector NADH similarly to wild-type PRK. For D42E and D42C, V(max) exhibits substantial decreases of 135- and 220-fold, respectively. Comparable substitutions for D169 result in smaller effects; D169E and D169C exhibit decreases in V(max) of 39- and 26-fold, respectively. Thus, regardless of the type of substitution, changes at D42 more profoundly affect catalytic rate than do comparable changes at D169. Precedent with enzymes in which cysteine replaces an acidic residue suggests that oxidation of the thiolate to a sulfinate can convert low-activity cysteine mutants into enzymes with improved activity. Periodate oxidation of cysteine-free PRK results in a slight decrease in activity. In contrast, comparable treatment of D42C and D169C proteins increases activity by 5- and 7-fold, respectively. Thus, for reasonably efficient catalysis, PRK requires anionic character in the side chains of residues 42 and 169. The enzyme can, however, tolerate substantial structural and chemical variability at these residues.

Phosphoribulokinase: Mutagenesis of the Mobile Lid and “P-Loop”

Phosphoribulokinase (PRK) catalyzes the in-line transfer of ATP’s yphosphoryl to the Cl hydroxyl of ribulose 5-phosphate (Ru5P) forming ribulose 1,5-bisphosphate, the Calvin cycle CO2 acceptor. Recently several acidic residues crucial to catalysis, D42 and D169, were identified in R. spheroides PRK (1). The mutation of these two residues to alanines profoundly reduced catalysis, with a 105-fold diminution in rate for D42 and a 104-fold diminution in rate for D169; E131 also has a significant influence on the catalytic efficiency. These acidic amino acids could function as activator cation ligands or as a catalytic base. A specific function in sugar phosphate substrate binding has been proposed for prokaryotic PRK’s R49, based on the large Km Ru5P, effect that is observed upon mutagenesis of this residue to glutamine (2).

Phosphoribulokinase: 3-Dimensional Structure & Catalytic Mechanism

Phosphoribulokinase (PRK) catalyzes production of the Calvin cycle’s CO2 acceptor, ribulose 1,5-bisphosphate. As might be anticipated for a reaction so crucial to carbon assimilation, PRK is highly regulated in both the prokaryotic and eukaryotic organisms in which it functions. In eukaryotes, activity is modulated by a thioredoxin-mediated thiol/disulfide exchange. The prokaryotic enzyme is allosterically regulated, with NADH functioning as a positive effector while AMP and PEP function as negative effectors.