Human serum albumin (HSA) is the most abundant protein in the circulatory system. Its principal function is to transport fatty acids, but it is also capable of binding a great variety of metabolites and drugs. Despite intensive efforts, the detailed structural basis of fatty acid binding to HSA has remained elusive. We have now determined the crystal structure of HSA complexed with five molecules of myristate at 2.5 A resolution. The fatty acid molecules bind in long, hydrophobic pockets capped by polar side chains, many of which are basic. These pockets are distributed asymmetrically throughout the HSA molecule, despite its symmetrical repeating domain structure.

tase genes have revealed a conserved and ordered modular 4 Corresponding author organization. Each module encodes a functional building unit containing~1000 amino acids, which specifically The non-ribosomal synthesis of the cyclic peptide recognizes a single amino acid. Within such a protein antibiotic gramicidin S is accomplished by two large template-directed peptide biosynthesis, the occurrence and multifunctional enzymes, the peptide synthetases 1 and specific order of the modules in the genomic DNA dictate 2. The enzyme complex contains five conserved subunits the number and sequence of the amino acids to be of~60 kDa which carry out ATP-dependent activation incorporated into the resulting oligopeptide. The modular of specific amino acids and share extensive regions of arrangement of peptide synthetases closely parallels the sequence similarity with adenylating enzymes such multienzyme complexes responsible for the biogenesis of as firefly luciferases and acyl-CoA ligases. We have fatty acids and of the polyketide family of natural products. determined the crystal structure of the N-terminal Furthermore, peptide synthetases, fatty-acid synthetases adenylation subunit in a complex with AMP and and polyketide synthetases all use enzyme-bound phospho-L-phenylalanine to 1.9 Å resolution. The 556 amino pantetheine cofactors as acyl carriers, in a thiotemplate acid residue fragment is folded into two domains with mechanism first proposed by Lipmann more than 20 the active site situated at their interface. Each domain years ago (Lipmann, 1971) and revised recently (Stein of the enzyme has a similar topology to the correspond et al., 1996). ing domain of unliganded firefly luciferase, but aIn particular, the synthesis of the cyclic antibiotic remarkable relative domain rotation of 94°occurs.gramicidin S has been studied in detail. Gramicidin S is This conformation places the absolutely conserved produced by the Gram-positive bacterium Bacillus brevis Lys517 in a position to form electrostatic interactions and consists of two identical pentapeptides joined head with both ligands. The AMP is bound with the phosto tail. It is synthesized by the multienzyme complex phate moiety interacting with Lys517 and the hydroxyl gramicidin S synthetase, which is encoded by the 19 kb groups of the ribose forming hydrogen bonds with grs operon that includes the genes grsA, grsB and grsT. Asp413. The phenylalanine substrate binds in a hydro-The grsT gene, which is located at the 5Ј-end of the grs phobic pocket with the carboxylate group interacting operon, encodes a 29 kDa protein homologous to fattywith Lys517 and the α-amino group with Asp235. The acid thioesterases. The grsA gene product, gramicidin S structure reveals the role of the invariant residues synthetase 1 (GrsA) is a protein composed of 1098 amino within the superfamily of adenylate-forming enzymes acids (Hori et al., 1989; Krätzschmar et al., 1989). and indicates a conserved mechanism of nucleotide GrsA activates L-phenylalanine to the corresponding acylbinding and ...

The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy by only 0.5-1.5 kcal mol-1. But the presence of an unpaired and charged donor or acceptor weakens binding by a further approximately 3 kcal mol-1.

Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.

The 3.3-A resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme to surround completely the DNA substrate, thereby allowing processive nucleotide polymerization without enzyme dissociation.