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Streptomyces olivaceoviridis secretes a so far unique protein of 18.7 kDa (CHB1) which lacks catalytic activity. It interacts highly specifically with α-chitin, but not with β-chitin, chitosan, or cellulose. Each of the five codons for tryptophan (Trp) in the chb1 gene was replaced by those for leucine (Leu) or tyrosine (Tyr). Eight corresponding mutant proteins and the wild-type protein were purified to homogeneity and their binding capacity to α-chitin was determined. The relative affinities to anti-CHB1 antibodies, the kinetics of binding, the dissociation constants, circular dichroism, and fluorescence emission spectra for three mutant types were compared to the characteristics of CHB1. The presented data lead to the following conclusions. (a) CHB1 presents a highly flexible protein lacking α-helices. (b) Replacement of each of the buried Trp residues (Trp134 and Trp184) leads to conformational alterations and, in due course, to a considerably reduced binding affinity of the protein. (c) The exchange of the exposed Trp 57 by either Leu or Tyr results in relatively slight topological changes, but entails a loss of binding capacity of about 90%. (d) The dissociation constant was highest for the mutant protein [L57]CHB1 (2.17 µM), followed by [LI34]CHB1 (0.91 µM) and [LI84]CHB1 (0.26 µM), and lowest for the progenitor CHB1 (0.11 µM), indicating its strong affinity to the unsoiuble substrate. (e) The data suggest that the exposed Trp57 contributes directly and significantly to the interaction of CHB1 with α-chitin. [ABSTRACT FROM AUTHOR]

Abstract: Gamma-butyrobetaine hydroxylase (GBBH) is a 2-ketoglutarate-dependent dioxygenase that catalyzes the biosynthesis of l-carnitine by hydroxylation of gamma-butyrobetaine (GBB). l-carnitine is required for the transport of long-chain fatty acids into mitochondria for generating metabolic energy. The only known synthetic inhibitor of GBBH is mildronate (3-(2,2,2-trimethylhydrazinium) propionate dihydrate), which is a non-hydroxylatable analog of GBB. To aid in the discovery of novel GBBH inhibitors by rational drug design, we have solved the three-dimensional structure of recombinant human GBBH at 2.0Å resolution. The GBBH monomer consists of a catalytic double-stranded β-helix (DBSH) domain, which is found in all 2KG oxygenases, and a smaller N-terminal domain. Extensive interactions between two monomers confirm earlier observations that GBBH is dimeric in its biological state. Although many 2KG oxygenases are multimeric, the dimerization interface of GBBH is very different from that of related enzymes. The N-terminal domain of GBBH has a similar fold to the DUF971 superfamily, which consists of several short bacterial proteins with unknown function. The N-terminal domain has a bound Zn ion, which is coordinated by three cysteines and one histidine. Although several other 2KG oxygenases with known structures have more than one domain, none of them resemble the N-terminal domain of GBBH. The N-terminal domain may facilitate dimer formation, but its precise biological role remains to be discovered. The active site of the catalytic domain of GBBH is similar to that of other 2KG oxygenases, and Fe(II)-binding residues form a conserved His–X–Asp–X n –His triad, which is found in all related enzymes. [ABSTRACT FROM AUTHOR]