Biomedical subjects

Michael T Boyne

Publications and source records attributed to Michael T Boyne.

5 recordsLinked to original sources

PubMed2007

Characterization of the aminocarboxycyclopropane-forming enzyme CmaC.

The biosynthesis of the coronamic acid fragment of the pseudomonal phytotoxin coronatine involves construction of the cyclopropane ring from a gamma-chloro-L-allo-Ile intermediate while covalently tethered as a phosphopantetheinyl thioester to the carrier protein CmaD. The cyclopropane-forming catalyst is CmaC, catalyzing an intramolecular displacement of the gamma-Cl group by the alpha carbon. CmaC can be isolated as a Zn2+ protein with about 10-fold higher activity over the apo form. CmaC will not cyclize free gamma-chloro amino acids or their S-N-acetylcysteamine (NAC) thioester derivatives but will recognize some other carrier protein scaffolds. Turnover numbers of 5 min-1 are observed for Zn-CmaC, acting on gamma-chloro-L-aminobutyryl-S-CmaD, generating 1-aminocyclopropane-1-carbonyl (ACC)-S-CmaD. Products were detected either while still tethered to the phosphopantetheinyl prosthetic arm by mass spectrometry or after thioesterase-mediated release and derivatization of the free amino acid. In D2O, CmaC catalyzed exchange of one deuterium into the aminobutyryl moiety of the gamma-Cl-aminoacyl-S-CmaD, whereas the product ACC-S-CmaD lacked the deuterium, consistent with a competition for a gamma-Cl-aminobutyryl alpha-carbanion between reprotonation and cyclization. CmaC-mediated cyclization yielded solely ACC, resulting from C-C bond formation and no azetidine carboxylate from an alternate N-C cyclization. CmaC could cyclize gamma,gamma-dichloroaminobutyryl to the Cl-ACC product but did not cyclize delta- or epsilon-chloroaminoacyl-S-CmaD substrates.

Amino Acids↗

PubMed2006

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units.

Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.

Acyl Carrier Protein↗

PubMed2006

Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster.

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.

Amino Acid Sequence↗

PubMed2006

Precise characterization of human histones in the H2A gene family by top down mass spectrometry.

Top Down analysis revealed that at least fourteen genes encoding histone H2A are coexpressed in HeLa cells. Characterization of these species revealed that all except H2A.Z and H2A.F/Z were alpha-N-acetylated, H2A.O and H2A.C,D,I,N,P were the most abundant, and those exceeding approximately 10% abundance lacked post-translational modifications. This unequivocal identification of H2A forms illustrates the advantages of Top Down Mass Spectrometry and provides a global perspective of H2A regulation through the cell cycle.

Acetylation↗

PubMed2005

Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry.

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Such biological variation compounded by the high sequence identity within gene families currently overwhelms the complete and routine characterization of mammalian proteins by MS. A new data base of human proteins (and their possible variants) was created and searched using tandem mass spectrometric data from intact proteins. This first application of top down MS/MS to wild-type human proteins demonstrates both gene-specific identification and the unambiguous characterization of multifaceted mass shifts (Deltam values). Such Deltam values found from the precise identification of 45 protein forms from HeLa cells reveal 34 coding single nucleotide polymorphisms, two protein forms from alternative splicing, and 12 diverse modifications (not including simple N-terminal processing), including a previously unknown phosphorylation at 10% occupancy. Automated protein identification was achieved with a median expectation value of 10(-13) and often occurred simultaneously with dissection of diverse sources of protein variability as they occur in combination. Top down MS therefore has a bright future for enabling precise annotation of gene products expressed from the human genome by non-mass spectrometrists.

Alternative Splicing↗