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Zhihao Zhuang

Publications and source records attributed to Zhihao Zhuang.

17 recordsLinked to original sources
PubMed2006

Structure-activity analysis of base and enzyme-catalyzed 4-hydroxybenzoyl coenzyme A hydrolysis.

In this study, the second-order rate constant k2 of base-catalyzed hydrolysis and the values of kcat, Km and kcat/Km of wild-type Pseudomonas sp. CBS3 4-hydroxybenzoyl coenzyme A (4-HBA-CoA) thioesterase-catalyzed hydrolysis of 4-HBA-CoA and its para-substituted analogs were measured. For the base-catalyzed hydrolysis, the plot of logk2 vs the sigma value of the para-substituents was linear with a slope (rho) of 1.5. In the case of the enzyme-catalyzed hydrolysis, the kcat/Km values measured for the para-substituted analogs defined substrate specificity. Asp32 was shown to play a key role in substrate recognition, and in particular, in the discrimination between the targeted substrate and other cellular benzoyl-CoA thioesters.

Acyl Coenzyme A↗
PubMed2006

An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex.

In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.

Adenosine Diphosphate↗
PubMed2006

Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation.

In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the subsequent binding of polymerase to the loaded clamp. Recently published evidence suggests that holoenzyme assembly in the T4 replisome may take place via more than one pathway [Zhuang, Z., Berdis, A. J., and Benkovic, S. J. (2006) Biochemistry 45, 7976-7989]. To demonstrate unequivocally whether there are multiple pathways leading to the formation of a productive holoenzyme, single-molecule fluorescence microscopy has been used to study the potential clamp loading and holoenzyme assembly pathways on a single-molecule DNA substrate. The results obtained reveal four pathways that foster the formation of a functional holoenzyme on DNA: (1) clamp loader-clamp complex binding to DNA followed by polymerase, (2) clamp loader binding to DNA followed by clamp and then polymerase, (3) clamp binding to DNA followed by clamp loader and then polymerase, and (4) polymerase binding to DNA followed by the clamp loader-clamp complex. In all cases, MgATP is required. The possible physiological significance of the various assembly pathways is discussed in the context of replication initiation and lagging strand synthesis during various stages of T4 phage replication.

Bacteriophage T4↗
PubMed2006

The structure of a ring-opened proliferating cell nuclear antigen-replication factor C complex revealed by fluorescence energy transfer.

Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp-RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATPgammaS cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.

Adenosine Triphosphate↗
PubMed2006

Structure, function, and mechanism of the phenylacetate pathway hot dog-fold thioesterase PaaI.

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins.

Binding Sites↗
PubMed2005

Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome.

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is "unlocked" by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.

Bacteriophage T4↗
PubMed2005

The oligomeric T4 primase is the functional form during replication.

Replisome DNA primases are responsible for the synthesis of short RNA primers required for the initiation of repetitive Okazaki fragment synthesis on the lagging strand during DNA replication. In bacteriophage T4, the primase (gp61) interacts with the helicase (gp41) to form the primosome complex, an interaction that greatly stimulates the priming activity of gp61. Because gp41 is hexameric, a question arises as to whether gp61 also forms a hexameric structure during replication. Several results from this study support such a structure. Titration of the primase/single-stranded DNA binding followed by fluorescence anisotropy implicated a 6:1 stoichiometry. The observed rate constant, k(cat), for priming was found to increase with the primase concentration, implicating an oligomeric form of the primase as the major functional species. The generation of hetero-oligomeric populations of the hexameric primase by controlled mixing of wild type and an inactive mutant primase confirmed the oligomeric nature of the most active primase form. Mutant primases defective in either the N- or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits. Collectively, these results provide strong evidence for the functional oligomerization of gp61. The potential roles of gp61 oligomerization during lagging strand synthesis are discussed.

Bacteriophage T4↗
PubMed2005

Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly.

The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and lagging-strand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the lagging-strand holoenzyme can occur without leading-strand DNA replication.

Bacteriophage T4↗
PubMed2004

The dynamic processivity of the T4 DNA polymerase during replication.

The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of approximately 0.0013 sec(-1) was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution/replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.

Bacteriophage T4↗
PubMed2004

The BH1999 protein of Bacillus halodurans C-125 is gentisyl-coenzyme A thioesterase.

In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a k(cat)/K(m) of 1.6 x 10(6) M(-1) s(-1) and the hydrolysis of 3-hydroxybenzoyl-CoA with a k(cat)/K(m) of 3.0 x 10(5) M(-1) s(-1). All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.

Amino Acid Sequence↗
PubMed2003

The structure of 4-hydroxybenzoyl-CoA thioesterase from arthrobacter sp. strain SU.

The 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a thioesterase. Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU. The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called "hot dog fold" composed of six strands of anti-parallel beta-sheet flanked on one side by a rather long alpha-helix. The dimers come together to form the tetramer with their alpha-helices facing outwards. This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their alpha-helices projecting toward the interfacial region. In the Arthrobacter thioesterase, each of the four active sites is formed by three of the subunits of the tetramer. On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter thioesterase. Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme. Although these two bacterial thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different.

Arthrobacter↗
PubMed2003

Characterization of the 4-hydroxybenzoyl-coenzyme A thioesterase from Arthrobacter sp. strain SU.

The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k(cat) of 6.7 s(-1) and a K(m) of 1.2 micro M. The k(cat) pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.

Amino Acid Sequence↗
PubMed2002

Kinetic, Raman, NMR, and site-directed mutagenesis studies of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase active site.

4-Hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase catalyzes the hydrolysis of 4-HBA-CoA to 4-hydroxybenzoate and CoA. X-ray crystallographic analysis of the liganded enzyme has shown that the benzoyl thioester and pantetheine moieties of the substrate ligand are bound in a narrow crevice while the nucleotide moiety rests on the protein surface (Thoden, J. B., Holden, H. M., Zhuang, Z. and Dunaway-Mariano, D. (2002) X-ray Crystallographic Analyses of Inhibitor and Substrate Complexes of Wild-type and Mutant 4-Hydroxybenzoyl-CoA Thioesterase, J. Biol. Chem., in press). Asp17 is positioned in the crevice, close to the substrate thioester C=O, which in turn interacts with the positive pole of an alpha-helix macrodipole. In this paper we report the results from spectral, mutagenesis, and kinetic studies which show (1) that substrate activation involves restricted thioester C=O conformational freedom and a modest enhancement of C=O bond polarization, (2) that the nucleotide unit of the substrate is bound through interaction with the protein surface, and (3) that Asp17 contributes a rate factor of 10(4), consistent with its proposed role of general base or nucleophile.

Adenine↗
PubMed2002

X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase.

The metabolic pathway by which 4-chlorobenzoate is degraded to 4-hydroxybenzoate in the soil-dwelling microbe Pseudomonas sp. strain CBS-3 consists of three enzymes including 4-hydroxybenzoyl-CoA thioesterase. The structure of the unbound form of this thioesterase has been shown to contain the so-called "hot dog" fold with a large helix packed against a five-stranded anti-parallel beta-sheet. To address the manner in which the enzyme accommodates the substrate within the active site, two inhibitors have been synthesized, namely 4-hydroxyphenacyl-CoA and 4-hydroxybenzyl-CoA. Here we describe the structural analyses of the enzyme complexed with these two inhibitors determined and refined to 1.5 and 1.8 A resolution, respectively. These studies indicate that only one protein side chain, Ser(91), participates directly in ligand binding. All of the other interactions between the protein and the inhibitors are mediated through backbone peptidic NH groups, carbonyl oxygens, and/or solvents. The structures of the enzyme-inhibitor complexes suggest that both a hydrogen bond and the positive end of a helix dipole moment serve to polarize the electrons away from the carbonyl carbon of the acyl group, thereby making it more susceptible to nucleophilic attack. Additionally, these studies demonstrate that the carboxylate group of Asp(17) is approximately 3.2 A from the carbonyl carbon of the acyl group. To address the role of Asp(17), the structure of the site-directed mutant protein D17N with bound substrate has also been determined. Taken together, these investigations suggest that the reaction mechanism may proceed through an acyl enzyme intermediate.

Aspartic Acid↗
PubMed2002

The YbgC protein encoded by the ybgC gene of the tol-pal gene cluster of Haemophilus influenzae catalyzes acyl-coenzyme A thioester hydrolysis.

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol-pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl-CoA and acyl-N-acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl-CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.

Acyl Coenzyme A↗