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by Ching-man Amy, Chau. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 142-147.

Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) to 2-hydroxypenta-2,4-dienoate (HPD) and benzoate. Although MCP hydrolases contain the catalytic triad (Ser112-His265-Asp237) and structural fold of the α/β-hydrolase superfamily, previous studies suggest they deviate from the classical hydrolytic mechanism in two respects: (1) enol-keto tautomerization precedes hydrolysis and (2) hydrolysis involves a gem-diol intermediate. Stopped-flow kinetic studies revealed rapid accumulation of a transient intermediate possessing a red-shifted absorption spectrum (λmax = 492 nm) versus HOPDA (λmax = 434 nm), consistent with an enzyme-bound, strained enolate (E:Sse). In studies with BphDLB400 variants, S112A trapped the E:Sse intermediate, implying that Ser112 is required for subsequent tautomerization and hydrolysis. His265 is required for E:Sse formation, as H265A variants instead generated a species assigned to a non-strained HOPDA enolate, which was not spectroscopically observed in the WT enzyme. The proposed importance of double bond strain in the reaction was supported by crystallographic observation of a non-planar, strained substrate in the S112A:HOPDA complex. Inhibition of BphDLB400 by 3-Cl HOPDA was investigated to understand a block in the degradation of polychlorinated biphenyls. BphDLB400 preferentially hydrolyzed 3-substituted HOPDAs in the order H > F > Cl > Me, indicating that steric bulk impairs catalysis. Kinetic analyses further indicated that large 3-substituents impede formation of the strained enolate by binding in an alternate conformation, as observed in the S112A:3-Cl HOPDA crystal structure. Finally, rate-determining hydrolysis of a benzoyl-enzyme was suggested from the observations that: (i) HOPDA and p-nitrophenyl benzoate were transformed with similar kcat values and (ii) yielded a common product ratio in the presence of methanol. Overall, the studies demonstrate the importance of an intermediate possessing significant double bond strain in an MCP hydrolase, establish the role of the catalytic His in forming this intermediate, indicate a mechanism of inhibition, and suggest the possibility that hydrolysis may proceed via an acyl-enzyme.

D-Desosamine, a 3-(dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin, neomethymycin, pikromycin, and narbomycin produced by Streptomyces venezuelae, plays an essential role in conferring biological activities to its parent aglycones. The proteins encoded by the desI and desII genes in the methymycin/pikromycin biosynthetic gene cluster have been proposed to catalyze C-4 deoxygenation in D-desosamine biosynthesis. DesI is a pyridoxal 5'-phosphate-dependent C4-aminotransferase and catalyzes a transamination reaction converting thymidine diphosphate (TDP)-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-glucose. DesII, which contains a [4Fe-4S] cluster binding motif, CXXXCXXC, has been identified as a member of the radical S-adenosylmethionine (SAM) enzyme superfamily by sequence analysis. To study the catalytic function of DesII, the desII gene was heterologously overexpressed in Escherichia coli and the DesII protein was purified to near homogeneity. Biochemical studies clearly established that the substrate for DesII is TDP-4-amino-4,6-dideoxy-D-glucose, and DesI and DesII function independently to carry out C-4 deoxygenation. DesII requires a [4Fe-4S]¹⁺ center and Sadenosylmethionine for activity. Accordingly, the originally proposed mechanism for C-4 deoxygenation in which DesI and DesII function together was revised. Two possible mechanisms have subsequently been proposed for the DesII reaction. The DesII catalysis is likely initiated by the formation of a 5'-deoxyadenosyl radical followed by the C-3 hydrogen atom abstraction. In the first possible route, the key step is a radical-induced deamination followed by the readdition of ammonia to the resulting cation radical intermediate, which is effectively a 1,2-amino shift, to form an aminol radical. Alternatively, the reaction may involve deprotonation of the 3-hydroxyl group to yield a ketyl radical anion to facilitate the [beta]-elimination of the ammonia group. Interestingly, DesII is flexible towards TDP-D-quinovose and TDP-3-amino-3,6-dideoxy-D-glucose. A possible biological reducing system, flavodoxin, flavodoxin reductase, and NADPH, for the reduction of the [4Fe-4S]²⁺ cluster, was also identified. Deuterium incorporation into SAM using C-3 deuterium-labeled substrate provides solid evidence for C-3 hydrogen atom abstraction by the 5'-deoxyadenosyl radical in the proposed mechanism. TDP-3-fluoro-3,6-dideoxy-D-glucose serves as a competitive inhibitor for DesII, which is in favor of deprotonation of the C-3 hydroxyl group being involved in DesII catalysis. / text

Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) to 2-hydroxypenta-2,4-dienoate (HPD) and benzoate. Although MCP hydrolases contain the catalytic triad (Ser112-His265-Asp237) and structural fold of the α/β-hydrolase superfamily, previous studies suggest they deviate from the classical hydrolytic mechanism in two respects: (1) enol-keto tautomerization precedes hydrolysis and (2) hydrolysis involves a gem-diol intermediate. Stopped-flow kinetic studies revealed rapid accumulation of a transient intermediate possessing a red-shifted absorption spectrum (λmax = 492 nm) versus HOPDA (λmax = 434 nm), consistent with an enzyme-bound, strained enolate (E:Sse). In studies with BphDLB400 variants, S112A trapped the E:Sse intermediate, implying that Ser112 is required for subsequent tautomerization and hydrolysis. His265 is required for E:Sse formation, as H265A variants instead generated a species assigned to a non-strained HOPDA enolate, which was not spectroscopically observed in the WT enzyme. The proposed importance of double bond strain in the reaction was supported by crystallographic observation of a non-planar, strained substrate in the S112A:HOPDA complex. Inhibition of BphDLB400 by 3-Cl HOPDA was investigated to understand a block in the degradation of polychlorinated biphenyls. BphDLB400 preferentially hydrolyzed 3-substituted HOPDAs in the order H > F > Cl > Me, indicating that steric bulk impairs catalysis. Kinetic analyses further indicated that large 3-substituents impede formation of the strained enolate by binding in an alternate conformation, as observed in the S112A:3-Cl HOPDA crystal structure. Finally, rate-determining hydrolysis of a benzoyl-enzyme was suggested from the observations that: (i) HOPDA and p-nitrophenyl benzoate were transformed with similar kcat values and (ii) yielded a common product ratio in the presence of methanol. Overall, the studies demonstrate the importance of an intermediate possessing significant double bond strain in an MCP hydrolase, establish the role of the catalytic His in forming this intermediate, indicate a mechanism of inhibition, and suggest the possibility that hydrolysis may proceed via an acyl-enzyme.