The focus of this paper is the hotdog-fold thioesterase THEM2 from human (hTHEM2; Swiss-Prot entry Q9NPJ3). In an earlier communication (Cheng, Z., Song, F., Shan, X., Wei, Z., Wang, Y., Dunaway-Mariano, D., and Gong, W. (2006) Crystal structure of human thioesterase superfamily member 2, Biochem Biophys Res Commun 349, 172-177.) we reported the apo crystal structure of hTHEM2. Herein, we report the results of an extensive hTHEM2 substrate screen, the structure determination of hTHEM2 complexed with the inert substrate analog undecan-2-one-CoA (in which O=C-CH2-S substitutes for O=C-S) and the kinetic analysis of active site mutants. The work described in this paper represents the first reported structure-function based analysis of a human hotdog-fold thioesterase. The catalytic mechanism proposed involves the Asp65/Ser83 assisted attack of a water molecule at the Gly57/Asn50 polarized thioester C=O and the Asn50 assisted departure of the thiolate leaving group. Thioesterase activity was observed with acyl-CoAs but not with the human acyl-ACP or with an acyl-Cys peptide. The medium-to-long chain fatty acyl-CoAs displayed the smallest Km values. The substrate specificity profile was analyzed within the context of the liganded enzyme to define the structural determinants of substrate recognition. Based on the results of this structure-function analysis we hypothesize that the physiological role of hHTEM2 involves catalysis of the hydrolysis of cytosolic medium-to-long chain acyl-CoA thioesters.
Studies of recombination-dependent replication (RDR) in the T4system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3 ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop͞replication fork. This partitioning mechanism enforced by the T4 recombination͞replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX͞uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative ''bubble migration'' mode of RDR could play a role in lesion bypass by the T4 replication machinery. Bacteriophage T4 provides an excellent model system for biochemical and genetic studies of recombinationdependent replication (RDR), because DNA replication and recombination are closely coupled throughout much of the phage life cycle. After infecting a host Escherichia coli cell, T4 first replicates its genome via an origin-dependent replication initiation pathway. This pathway is shut off after a few rounds of replication, after expression of the T4 uvsW RNA͞DNA helicase, which resolves R loops required for origin function (1). T4 then relies on a recombination-dependent mechanism to initiate DNA synthesis, and this pathway accounts for a large fraction of the total DNA synthesis observed during T4 infection. In the T4 RDR pathway (reviewed in refs. 2 and 3), branched recombination intermediates generated by the phage homologous recombination machinery are captured and converted into semiconservative DNA replication forks. T4 RDR requires all of the major phage-encoded DNA replication and recombination enzymes including: gp43 (DNA polymerase), gp45 (sliding clamp), gp44͞62 (clamp loader), gp32 [single-stranded DNA (ssDNA) binding protein or ssb], gp61 (primase), gp41 (DNA helicase), gp59 (helicase loader; replication mediator protein or RMP...
A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product -catechol 2,3-dioxygenase -in Methylobacterium organophilum XX. The specific activity of the enzyme was four times higher in methanol-grown M. organophilum XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M. organophilum XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria : Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the xyZE gene were mobilized into Mox-mutants of M. organophilum XX and M. extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.
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