Substrate Specificity Switch Research Articles

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structure-based mechanism of conversion.

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (M(r), 290,000) bovine milk XDH at 2.1-A resolution and XO at 2.5-A resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching.

We have investigated the properties of the cytoplasmic domain (FlhB(C)) of the 383-amino-acid Salmonella membrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod- and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhB(C) was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhB(CN) and FlhB(CC), which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhB(delta CC) (i.e., the N-terminal transmembrane domain FlhB(TM) plus FlhB(CN)) and FlhB(CC), resulted in restoration of both motility and flagellar protein export to an flhB mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhB(C), existing as a FlhB(CN)-FlhB(CC) complex on an affinity blot membrane, bound the rod- and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhB(C) (mutant or wild type) or the FlhB(CC) polypeptide alone bound FlgD and FliC to about the same extent. FlhB(CN) by itself did not bind substrates appreciably. We propose that FlhB(C) has two substrate specificity states and that a conformational change, mediated by the interaction between FlhB(CN) and FlhB(CC), is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhB(C) resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.

Interactions among components of the Salmonella flagellar export apparatus and its substrates.

We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild-type cells and inhibition of the export of substrates such as the hook protein FlgE. Co-overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His-FLAG-tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook-type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament-type export substrates FlgK and FlgL (hook-filament junction proteins) and FliC (flagellin). We tested for protein-protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook-type substrates were strong, whereas those with filament-type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP-dependent process mediated by FliI.

FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly.

In wild-type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at approximately 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild-type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook-filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N-terminal region of FliK, but not by C-terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook-length control and the switching of export substrate specificity.

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.

Selective Modification of Alkylammonium Ion Specificity in Trimethylamine Dehydrogenase by the Rational Engineering of Cation-π Bonding

In trimethylamine dehydrogenase (TMADH), substrate is bound in the active site by organic cation-pi bonding mediated by residues Tyr-60, Trp-264, and Trp-355. In the closely related dimethylamine dehydrogenase (DMADH), modeling suggests that a mixture of cation-pi bonding and conventional hydrogen bonding is responsible for binding dimethylamine. The active sites of both enzymes are highly conserved, but three changes in amino acid identity (residues Tyr-60 --> Gln, Ser-74 --> Thr, and Trp-105 --> Phe, TMADH numbering) were identified as probable determinants for tertiary --> secondary alkylammonium ion specificity. In an attempt to switch the substrate specificity of TMADH so that the enzyme operates more efficiently with dimethylamine, three mutant proteins of TMADH were isolated. The mutant forms contained either a single mutation (Y60Q), double mutation (Y60Q x S74T) or triple mutation (Y60Q x S74T x W105F). A kinetic analysis in the steady state with trimethylamine and dimethylamine as substrate indicated that the specificity of the triple mutant was switched approximately 90,000-fold in favor of dimethylamine. The major component of this switch in specificity is a selective impairment of the catalytic efficiency of the enzyme with trimethylamine. Rapid-scanning and single wavelength stopped-flow spectroscopic studies revealed that the major effects of the mutations are on the rate of flavin reduction and the dissociation constant for substrate when trimethylamine is used as substrate. With dimethylamine as substrate, the rate constants for flavin reduction and the dissociation constants for substrate are not substantially affected in the mutant enzymes compared with wild-type TMADH. The results indicate a selective modification of the substrate-binding site in TMADH (that impairs catalysis with trimethylamine but not with dimethylamine) is responsible for the switch in substrate specificity displayed by the mutant enzymes.