Swiveling domain mechanism in pyruvate phosphate dikinase.

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.

Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): a cofactor bound at a site formed by a knot.

The crystal structures of YibK from Haemophilus influenzae (HI0766) have been determined with and without bound cofactor product S-adenosylhomocysteine (AdoHcy) at 1.7 and 2.0 A resolution, respectively. The molecule adopts an alpha/beta fold, with a topology that differs from that of the classical methyltransferases. Most notably, HI0766 contains a striking knot that forms the binding crevice for the cofactor. The knot formation is correlated with an alternative arrangement of the secondary structure units compared with the classical methyltransferases. Two loop regions undergo conformational changes upon AdoHcy binding. In contrast to the extended conformation of the cofactor seen in the classical methyltransferase structures, AdoHcy binds to HI0766 in a bent conformation. HI0766 and its close sequence relatives are all shorter versions of the more remotely related rRNA/tRNA methyltransferases of the spoU sequence family. We propose that the spoU sequence family contains the same core domain for cofactor binding as HI0766 but has an additional domain for substrate binding. The substrate-binding domain is absent in HI0766 sequence family and may be provided by another Haemophilus influenzae partner protein, which is yet to be identified.

A catalytic mechanism for D-Tyr-tRNATyr deacylase based on the crystal structure of Hemophilus influenzae HI0670.

D-Tyr-tRNA(Tyr) deacylase is an editing enzyme that removes d-tyrosine and other d-amino acids from charged tRNAs, thereby preventing incorrect incorporation of d-amino acids into proteins. A model for the catalytic mechanism of this enzyme is proposed based on the crystal structure of the enzyme from Haemophilus influenzae determined at a 1.64-A resolution. Structural comparison of this dimeric enzyme with the very similar structure of the enzyme from Escherichia coli together with sequence analyses indicate that the active site is located in the dimer interface within a depression that includes an invariant threonine residue, Thr-80. The active site contains an oxyanion hole formed by the main chain nitrogen atoms of Thr-80 and Phe-79 and the side chain amide group of the invariant Gln-78. The Michaelis complex between the enzyme and D-Tyr-tRNA was modeled assuming a nucleophilic attack on the carbonyl carbon of D-Tyr by the Thr-80 O(gamma) atom and a role for the oxyanion hole in stabilizing the negatively charged tetrahedral transition states. The model is consistent with all of the available data on substrate specificity. Based on this model, we propose a substrate-assisted acylation/deacylation-catalytic mechanism in which the amino group of the D-Tyr is deprotonated and serves as the general base.

From structure to function: YrbI from Haemophilus influenzae (HI1679) is a phosphatase.

The crystal structure of the YrbI protein from Haemophilus influenzae (HI1679) was determined at a 1.67-A resolution. The function of the protein had not been assigned previously, and it is annotated as hypothetical in sequence databases. The protein exhibits the alpha/beta-hydrolase fold (also termed the Rossmann fold) and resembles most closely the fold of the L-2-haloacid dehalogenase (HAD) superfamily. Following this observation, a detailed sequence analysis revealed remote homology to two members of the HAD superfamily, the P-domain of Ca(2+) ATPase and phosphoserine phosphatase. The 19-kDa chains of HI1679 form a tetramer both in solution and in the crystalline form. The four monomers are arranged in a ring such that four beta-hairpin loops, each inserted after the first beta-strand of the core alpha/beta-fold, form an eight-stranded barrel at the center of the assembly. Four active sites are located at the subunit interfaces. Each active site is occupied by a cobalt ion, a metal used for crystallization. The cobalt is octahedrally coordinated to two aspartate side-chains, a backbone oxygen, and three solvent molecules, indicating that the physiological metal may be magnesium. HI1679 hydrolyzes a number of phosphates, including 6-phosphogluconate and phosphotyrosine, suggesting that it functions as a phosphatase in vivo. The physiological substrate is yet to be identified; however the location of the gene on the yrb operon suggests involvement in sugar metabolism.

Pyruvate site of pyruvate phosphate dikinase: crystal structure of the enzyme-phosphonopyruvate complex, and mutant analysis.

Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, phosphonopyruvate (K(i) = 3 microM), have been obtained in the presence of Mg(2+). The structure has been determined and refined at 2.2 A resolution, revealing that the Mg(2+)-bound phosphonopyruvate binds in the alpha/beta-barrel's central channel, at the C-termini of the beta-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK-phosphonopyruvate complex and the structures of two complexes of pyruvate kinase, one with Mg(2+)-bound phospholactate and the other with Mg(2+)-oxalate and ATP, revealed that the two enzymes share some key features that facilitate common modes of substrate binding. There are also important structural differences; most notably, the machinery for acid/base catalysis is different.

Assisting functional assignment for hypothetical Heamophilus influenzae gene products through structural genomics.

The three-dimensional structures of Haemophilus influenzae proteins whose biological functions are unknown are being determined as part of a structural genomics project to ask whether structural information can assist in assigning the functions of proteins. The structures of the hypothetical proteins are being used to guide further studies and narrow the field of such studies for ultimately determining protein function. An outline of the structural genomics methodological approach is provided along with summaries of a number of completed and in progress crystallographic and NMR structure determinations. With more than twenty-five structures determined at this point and with many more in various stages of completion, the results are encouraging in that some level of functional understanding can be deduced from experimentally solved structures. In addition to aiding in functional assignment, this effort is identifying a number of possible new targets for drug development.